Pharmacodiagnosis Test Targeting Oncology and Neurodegeneration

ABSTRACT

The present invention relates to the field of medicine and biology. It concerns a new test for screening and therapeutic follow-up in oncology. More particularly, it relates to diagnostic and/or therapeutic tests in oncology and on neurodegenerative diseases. Molecular targeting by peptide vectors and antibodies or by small interfering RNAs (siRNAs) opens a new concept of interdependence for diagnostic and therapeutic tools.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation in part application of U.S. patentapplication Ser. No. 12/282,117 filed on Sep. 8, 2008. This applicationis also a continuation in part application of International PatentApplication Serial No. PCT/FR2011/000155 filed on Mar. 18, 2011.

FIELD OF THE INVENTION

The present invention relates to the field of medicine and biology. Itconcerns a new test for screening and therapeutic follow-up in oncology.More particularly, it relates to diagnostic and/or therapeutic tests inoncology and on neurodegenerative diseases. Molecular targeting bypeptide vectors and antibodies or by small interfering RNAs (siRNAs)opens a new concept of interdependence for diagnostic and therapeutictools.

The inventor highlights the mechanisms of molecular interactions and theinterest of biochips dedicated according to cancers with a multi-therapyadded with therapeutic additives to slow down the formation of theselesions.

The comprehension of the plays of balance between under-expression andover-expression of genes according to their localization in a cellularcompartment allows to open a field of finer differential analysis and toadapt a multi-therapy with siRNA and peptides by molecular targeting.

DESCRIPTION OF THE STATE OF THE ART

Age-related neurodegenerative diseases and cancers both involve amodification of the physiological process of programmed cell death orapoptosis. Neuronal death is abnormally accelerated duringneurodegenerative diseases such as Alzheimer's disease, Huntington'sdisease, Parkinson's disease etc. . . . On the other hand, thecancerization process corresponds to a blocking of apoptosis, whichresults in an uncontrolled multiplication of cells. The link betweenthese two processes has currently become a major field of investigationin research on aging. The control of the balance between cell division(mitosis), differentiation and programmed cell death (apoptosis), isfundamental during normal physiological processes, including embryonicdevelopment, tissue regeneration and aging. An impairment of thisbalance can lead to major pathological situations such as the formationof tumors or some neurodegenerative diseases.

Cancer is one of the principal causes of mortality throughout the world.While, over the course of the last generation, the percentages of deathsrelated to cardiac and cardiovascular diseases and a large number ofother diseases has decreased, the number of deaths related to thevarious forms of cancer has increased.

Despite the rapid advance in our understanding of the various forms ofcancer, the low survival rates can generally be attributed to inadequatediagnosis and inadequate treatment. Most tumors can only be detectedwhen they reach a size of approximately 1 cm. Since a continuouslydeveloping tumor takes a relatively short period of time to evolve to astage which is incompatible with survival, this leaves little time for atherapeutic intervention. Early diagnosis therefore becomes the key tosuccess for the treatment of cancer. Skin cancer for example, is themost widespread cancer in Canada. In 1992 alone, 50 300 new cases ofskin cancer were reported, compared with 19 300 cases of lung cancer, 16200 cases of colorectal cancer and 15 700 cases of breast cancer. Inother words, skin cancer is as common as the three main types of cancercombined. Its incidence continues to increase, with 64 200 new casesthereof in 1997, corresponding to an increase of 14 000 cases in 5years. In particular, the incidence of malignant melanoma is increasingat a rate of 2% per year. Early diagnosis remains the key to aneffective treatment. A malignant tumor is readily accessible and can beremoved with minor surgery. In fact, recovery is 100% if skin cancer isdetected early enough. Early diagnosis of skin cancer remains howeverdifficult . . . . It thus becomes important to be able to distinguishthese two types of skin cancers. A final diagnosis of skin cancerrequires a biopsy and a histological analysis. However, the decision tosend a biopsy for analysis (or even if a patient must be referred with adermatologist) becomes very subjective. There are several biopsies whichare not taken whereas they would have being.

Colon cancer is the third most common cause of cancer-related mortalityin men and women in North America (16 200 cases per year). Earlydetection, leading to an early intervention, has demonstrated thattreatment success and survival rate can be improved. For example, the5-year survival rate is 92% for a patient whose disease was detected atan early stage, whereas the rate drops to approximately 60% in patientswith a localized cancer, and to approximately 6% in those withmetastases. However, only a third of colon cancers are detected at anearly stage. One of the reasons for this delay in diagnosis is theabsence of a sensitive, relatively inexpensive and non-invasivescreening test. Breast cancer is one of the most common cancers inwomen, together with colon cancer. The mortality rate is the highestamong cancers affecting women. There are very few diagnostic markersallowing the detection of breast cancer and they only have a predictivevalue of 20%. There are no markers, either, which can detect ordetermine the invasiveness or the aggressiveness of metastatic cancercells or which permit therapeutic monitoring.

For a multitude of reasons, early diagnosis remains illusory for mostforms of cancer. For certain forms of cancer, disease-specific markersare not available or are only available at an advanced stage of thedisease, making diagnosis difficult. In other forms of cancer, themarkers are available but are not always specific for the disease orthey may be associated with its benign form.

A hope this year with the marker who is the receiver with the FSH ispossible. As one of our markers of the LIV21 complex (cf. figure) it iscommon to several cancers and allows an early diagnosis, it also appearsin the vessels as ours which one can see in Alexa revealed on the levelof the endothelial cells, but in more we observe a marking of theerythrocytes and also collagen. During the last years, considerableprogresses were made in the comprehension of the means implemented bythe oncogenes and tumor suppressor genes to control cell proliferationand the apoptosis. One of the main targets of these regulators is thefamily of E2F-type transcription factors in the E2F and RB proteinsignaling pathway.

In other cases still, the techniques exist but the prohibitory cost toin general implement them to the population makes them inappropriate.

These cancers may find it beneficial to be studied and diagnosed bybiochip including the bio-markers of the major pathways of regulation toallow an adjustment of the multi-therapy according to the results ofunder expression and over-expression of genes of the complex Liv21. Thusthe processing by standard molecular targeting siRNA or monoclonalantibodies combined or not with chemical therapeutic assets is of agreater effectiveness.

The neuroblastes are neuronal precursors resulting from the splitting ofthe neuro-epithelial cells. There are immature embryonic neurons whichcan still divide contrary to the mature neurons which cannot enter inmitosis.

The neuroblastoma, also called sympathoblastome, is a malignant tumordeveloped at the expense of the cells of the neural crest, which giverise to the sympathetic nervous system. It is of the solid tumor mostfrequent in the child (8 to 10% of cancers before 15 years) and inparticular about the very young child (average age 4 years with 90%which have less than 5 years). Its incidence is from 1 to 3 cases for100 000 older children from 0 to 14 years. There are no causes nor ofrecognized mailmen which support occurred of a neuroblastoma.

The neuroblastoma is discovered starting from symptoms due to theprimary neoplasm (mass tumoral, in particular abdominal, or compressionof a nearby part, such spinal-cord), to metastases or an endocrinesecretion (deterioration of the general state, diarrhea, arterialhypertension . . . ). The neuroblastoma can develop starting from anunspecified component of the system highly-strung person sympatheticnerve, generally at the abdominal level. At the time of the diagnosis,the tumor can be localized on the level of one only part, the local orregional level, or be from the disseminated start. The metastatic sitesmost frequent are the bone, bone marrow, the liver and the skin. Thus,the majority of the localized tumors have an excellent forecast. It isthe same for those of the children of less than one year, independentlyof the stage of the tumor. Some of these tumors regress evenspontaneously. On the contrary, approximately 60% of the children ofmore than one year forward a metastatic neuroblastoma from the start ofpoor prognosis.

The variety of the clinical presentation is in relation to theexpression of certain biochemical markers and molecular (DNA ploidy,amplification of the oncogene n-myc, Trk receptor expression, loss ofthe chromosome 1p, excess of the 17q . . . ). Thus, diffuse tumorsoccurring in the child of more than 1 year and on-expressing theoncogene myc are often chemo-resistant and have a poor prognosis(Berthold and Al, 1990; Schweigerer and Al, 1990). In the same way, theTrkB receptor expression and of BDNF allows a system of survivalautocrine neuroblastic cells and induces the neuritic growth. It is alsoassociated with the amplification of N-Myc and thus with a poorprognosis (Nakagawara and Al, 1994). On the contrary, the TrkA receptorexpression, which induces the differentiation of the neuroblastoma(Borrello and Al, 1993; Eggert and Al, 2000), and TrkC (Yamashiro andAl, 1996) are rather associated with a good forecast. Thus, it is commonto say that the TrkA receptor expression is conversely correlated withthe amplification of the oncogene n-myc.

During the development, the gliaux precursors give rise to theastrocytes, oligodendrocytes, microglial cells, choroidal cells andependymaires cells in the SNC, and with the Schwann cells in theperipheral nervous system. Thus, the cells gliales play a crucial rolein differentiation (Lemke, 2001) and the survival of the neurons (Bar,2000). These cells ensure the nutrition of the neurons, manage connectedthem inter-neuronal, control the neurotransmitters.

The glioblastomas are the malignant tumors astrocytaires (grade IVaccording to the classification of the World Health Organization) mostundifferentiated of SNC and they are generally found on the level of thecerebral hemispheres. In the adult, they are the most frequent braintumors (20% of all the intracranial tumors) with an angle of attack ofabout 3 new cases a year and for 100.000 inhabitants, that is to sayapproximately 2400 new cases a year in France. They occur at any age butin 70% of the cases between 45 and 70 years.

The glioblastomas form soft masses, rich in blood-vessels, from 3 to 10cm in diameter, of vinous color, heterogeneous with active compact areasand areas of necroses wide, strewn with vessels thromboses and whichinfiltrate brain tissue. However, these tumors are not practically everassociated with the appearance of metastases. There are surrounded by aedema which increases the suffering of the brain. Typically, they areexpressed by signs of intracranial hypertension which often joinschanges of the behavior, with crises comitiales, focal neurologicdeficits.

This tumor evolves quickly, in 2-3 month, and even after surgery,radiotherapy then chemotherapy, its forecast remains dark except if theglioblastoma comes from the tumor can be localized on the level of oneonly part, the local or regional level, or be from the disseminatedstart. The metastatic sites most frequent are the bone, bone marrow, theliver and the skin. Thus, the majority of the localized tumors have anexcellent forecast. It is the same for those of the children of lessthan one year, independently of the stage of the tumor. Some of thesetumors regress even spontaneously. On the contrary, approximately 60% ofthe children of more than one year forward a metastatic neuroblastomafrom the start of poor prognosis.

The variety of the clinical presentation is in relation to theexpression of certain biochemical markers and molecular (DNA ploidy,amplification of the oncogene n-myc, Trk receptor expression, loss ofthe chromosome 1p, excess of the 17q . . . ). Thus, diffuse tumorsoccurring in the child of more than 1 year and on-expressing theoncogene myc are often chemo-resistant and have a poor prognosis(Berthold and Al, 1990; Schweigerer and Al, 1990). In the same way, theTrkB receptor expression and of BDNF allows a system of survivalautocrine neuroblastic cells and induces the neuritic growth. It is alsoassociated with the amplification of N-Myc and thus with a poorprognosis (Nakagawara and Al, 1994). On the contrary, the TrkA receptorexpression, which induces the differentiation of the neuroblastomata(Borrello and Al, 1993; Eggert and Al, 2000), and TrkC (Yamashiro andAl, 1996) are rather associated with a good forecast. Thus, it is commonto say that the TrkA receptor expression is conversely correlated withthe amplification of the oncogene n-myc.

The receivers of the superfamily of TNF-R were studied in theneuroblastic cells. Thus, the Fas receiver could be highlighted at thelevel of certain neuroblastic cells (Gross et al., 2001), cells whichcan be (Barthlen et al., 1999; Riffkin et al., 2001) or not (Bian etal., 2004) resistant to the apoptosis induced by Fas. The sensitivity ofthe neuroblastic cells does not depend only on the Fas receptorexpression but on the presence or not of the caspase-8 in cells (Kisengeet al., 2003). The receptors of TRAIL are also expressed in theneuroblastic cells.

The neuroblastoma is one of most common in the child. Death rate is thehighest of all cancers assigning the children.

There are very few diagnostic markers able to detect the neuroblastoma.There are no either markers which can detect or determine the invasivityand the aggressiveness of the metastatic cancerous cells or which allowsa therapeutic follow-up.

During the last years, considerable progresses were made in thecomprehension of the means implemented by the oncogenes and tumorsuppressor genes to control cell proliferation and the apoptosis. One ofthe main targets of these regulators is the family of the mailmen oftranscription of the type E2Fs (E2F1, E2F2, E2F3, E2F4 etc. . . . ) inthe channel of indication of the proteins E2Fs and RB. These proteinsplay a central role in the control of cell division by coupling theregulation of genes necessary to the cell cycle progression with theextracellular signals (mitogenes, inhibitors of the proliferation). Itbehaves as an oncogene by stimulating tumor cell proliferation. Myc Nand Aurora kinase are also oncogenes implied in the proliferation of theneuroblastoma. E2F3 modulates the form of the RNA m of Aurora during thecell cycle. In addition, certain data suggest that the mailmen oftranscription E2F are critical for total activation and the repressionof Myc N in the neuroblastoma. The combination of the dosages of genesof Myc N and the Survivin by RTPCR is correlated at the stage of theclinical change of the neuroblastoma.

Among the expressed genes are found:

-   -   over-expression of the E2F4 transcription factor and the c-myc        oncogene which induce apoptosis of post-mitotic cells by        accumulation of oxygenated reactants (Tanaka, 2002) and the N        Myc which is amplified in 35% of the cases of neuroblastoma,        gene ALK, DDX1 and CRABP II too. HGMA1 and the Survivin also.        The repression of HGMA1 by RNA interference reduces the cell        proliferation of the neuroblastoma. N Myc induces the expression        of FAK. N Myc down reguls the expression mRNA of many genes        having a role in cellular architecture.    -   the gene p53, which belongs to the tumor suppressor gene family,        blocks the cell cycle in the case of DNA lesion. It has now been        demonstrated that this gene is also involved in the progression        of apoptosis (Oren, 1994; Yonish-Rouach, 1996);    -   the cyclin D1, one of the proteins constituting the regulatory        subunits of cell cycle kinases, which is essential for cell        cycle progression. This protein is also expressed during        apoptosis in various cell types (Han et al, 1996; Pardo et al,        1996).

A transcription factor Zinc finger can stop the activity of the D1cyclin and lock the cycle. Data suggest that the mailmen oftranscription E2F are critical for total activation and the repressionof MYCN in the neuroblastomas.

-   -   chk1 and 2, crb2, p21 and other oncogenes and cytokines such as        TNF alpha etc. . . . It would be of great interest to have novel        diagnostic methods detecting the presence of cancer with greater        specificity and making it possible to distinguish between        aggressive cancer cells with the tendency to metastasize and        those which are more localized and have a lower probability of        metastasizing. A marker capable of revealing cell proliferation        would therefore be of great use. The works of the professor Jean        Louis Mendel about the glial markers and in particular on the        GFAP and the NF70 advanced the diagnosis, the expression of the        synemine in the glial tumors is an important way of research        like the study of the mutations of the Ras/MAPK channel. The        deletion of chromosome 1 in the area 1p36 and the amplification        of gene MYC N sign the state of proliferation, a forecast of        neuroblastoma and an unfavourable histology. In 35% of the        cases, MYC N is amplified, in 58% of the cases it is chromosome        17 in q23 and in 35% of the cases chromosome 1 in p36 is        deleted.

Complex LIV21 will be studied by RT PCR and biochip and its cytoplasmicmarkers of interest and the membrane protein of the complex Liv 21 too.

21 additional markers and new sequences of complex LIV21 compared to thefirst and with the second patent deposited will allow a notableimprovement and an improvement of the pharmaco diagnostic tests which wepropose.

It is thanks to this improvement that the therapeutic adjustment inmulti-therapy will be able to allow a greater effectiveness ofprocessing and as it is thanks to this improvement as the diagnosis ofthe glial tumors will be done more precisely with a thorough knowledgeon the forecast, the grade and the development of these tumors. Anotherimprovement made it possible to have a larger precision in the study ofthe expression levels of various important genes for the diagnosis ofglial tumor, it is the troubleshoot of a standardization of the tissuesamples like punctures of the cerebrospinal fluid. Thus the comparisonsof profiles of expression are more reliable and undergo lessfluctuations due to skews of observation. All these new parameters whichdo not increase the number of variables but which decrease thebackground noise largely improve the diagnosis and the processing of thegliales tumors. ki 67 and Cafl are nuclear markers indicating theproliferation state of many cancers (Almouzny; Curie Institut). Liv21complex genes will be the cytoplasmic markers at least equal andcomplementary to the previously identified nuclear ones.

SUMMARY OF THE INVENTION

The present invention concerns new polypeptide, ribonuclotidic andnucleotidic sequences to integrate into a novel test for screening forreinduction of the cell cycle targeting oncology and the use of some ofthese same functionalized sequences, like auxiliary processing bymolecular targeting. It is about a major improvement without which thediagnosis and the test pharmaco diagnostic could be only partial. Theconsequence would be a less effective processing and of less broadimplementation according to the heterogeneity of cancers. It is thusabout a diagnostic test and a prognostic test for various cancers. Moreparticularly, the invention concerns the use of the genes or proteins ofthe Liv21 complex and of their derivatives as therapeutic tools or asdiagnostic and prognostic markers for cancers. The invention thereforeconcerns the detection of the LIV21 gene or LIV21 protein with a kitcomprising LIV21-specific antibodies or LIV21 specific probes. Thepresent invention also consist in using all the proliferation markersand transcription factors which play a role in the cancerization and insome cases, neurodegeneration process. The invention lies in the use ofquantitative RT PCR and the PCR (QPCR) twinned at the manufacture ofdiagnostic DNA biochips, proteins biochips and antibody arrays includingknown antibodies directed against various proteins of theLIV21-associated complex according to the phases of the cell cycle, thatis, without restriction thereto: peptides and antibodies specific forRBP2, E2F4, E2F1, SUMO, HDAC1, crb2, Int2, cmd2, cycE/cdk2, cdk1, CREB 1and p300, Rb, p107 and p130 of the pocket protein family. In addition,antibodies specific for NFkB, cdc2A, mdm2, p21, p53, p65, BRCA1,TNFalpha, TGF beta. The new sequences are the poly-nucleotidic andpolypeptide sequences of Liv21F, Liv21H, etc. . . . (list additional ofthe sequences: SEQ ID No 171 to 185) and the sequences of genes,proteins, and corresponding antibodies lately integrated in thepharmaco-diagnostic biochips are: Myc N, ALK, HMGA1, DDX1, GFAP, NF70,AD7cNTP, FAB3, Serin C2, Synemine, PDX1, HDAC6, TPX2, DKK1, DKK3, HUD,ID2, SKP2, TP531NP1, VEGF, NLRR1, PAX3-FKHR, NDP kinase A, Bora ?,Aurora A, Survivin

The protein arrays will make it possible to study the proteinicinteractions and the post traductional modifications, more particularlythe phosphorylations and methylations of certain proteins which sign astate characteristic of the sick cell different from the proteinicinteractions and metabolism of the healthy cell. The state of expressionand silencing of certain genes being different. The rate of expressionof each biomolecule in front of being observed distinctly in eachcellular compartment to be able in the second time controlled being(under expressed or on expressed by a standard auxiliary processingbiomolecule or siRNA or vectorized peptides).

The biochips with nucleotidic sequences will make it possible to studyin nuclear cellular extracts and in addition cytoplasmic or membran,under expressions or on gene expressions and the ratios between genes asbetween proteins of complex Liv21 and its associated partners, theanalysis of the interactions within the metabolic complexes is a key ofthe diagnosis and also passes by the study of the functional fields.

A first objective of the present invention is to demonstrate a methodfor the detection and prognosis of cancer and of its metastaticpotential which makes it possible to adjust a multitherapy targeted.Preferably, the cancer is selected from breast cancer on cerebralcancers and more particularly the glioblastoma, the neuroblatoma,without being limited thereto.

One aspect of the present invention consists of the use of LIV21 complexnew sequences as prognostic indicator for cancer and his therapeuticmonitoring.

Indeed, when Liv21 is localized in the cytoplasm, the cancer cells intissues are aggressive. Conversely, when the product of gene expressionLiv21F is preferentially localized in the cellular core, this is aprognostic indicator that the tissue cells are differentiated andquiescent and thus noninvasive. We define the Liv21 complex by theprotein extract and peptides studied by mass spectrometry such as Maldiand ESIMSMS or Maldi T of T of. The said extract has been obtained bybinding of the Liv21 complex to one of its polyclonal antibodiesdescribed in the patent (PCT/FR2006/000510).

The Liv21 complex is defined by its mass spectrometry global profile(FIG. 5) and the number and the molecular weight of protein extractsbands obtained on the acrylamide gels of FIGS. 1A and 1B as a functionof temperature at which the sample is submitted and of describedmigration conditions. In fact, when for example Liv21F peptide islocated in the cytoplasm and we reveal directly for example by in situhybridization or by biochip analysis its higher expression in thecytoplasm, the cancer cells in the tissues are aggressive. Conversely,when the LIV21 gene expression product or the expression of Liv21Fpeptide is preferentially located in the cell nucleus, this is aprognostic indicator that the cells of the tissue are differentiated andquiescent and therefore noninvasive. This observation is associatedunder investigation with expression, phosphorylation and localization ofthe other factors of complex Liv21 and with these partners ofinteraction. The effectiveness of a cancer treatment can also bemonitored by the traceability of new sequences and of these proteinsLiv21F and Liv21K, of this Liv21 protein complex, and of its derivativesand ratios with the associated proteins but also by Diagmicroarray andsensorchip including among others this protein and its Liv21 associatedcomplex (FIG. 12 and FIG. 14).

Moreover, detection of protein kinase C epsilon (PKCs) is alsoadvantageous since it has been determined that PKCs phosphorylates theLIV21 protein in order to maintain it in the cytoplasm. Thus, asignificant increase in PKCs is indicative of the presence of cancercells. Moreover, the LIV21/PKCS ratio increases in the cytoplasmicfraction of cancer cells. The same is true of the detection of HDAC1,which has been shown to be involved in PML/SUMO/Rb/HDAC-1. Moregenerally, the HDACs family plays a key role in the regulation of geneexpression, when the HDACs are overexpressed, they induce tumorsuppressor gene silencing, hence the advantage of using HDAC inhibitorsin therapy, combined with other inhibitors which regulate the metabolicpathway involving the protein complex which contains Liv21.

In addition, the detection of the E2F1, E2F2, E2F3 and/or E2F4 proteinsis advantageous. In fact, the LIV21 protein forms a complex with E2F4,which is capable of inhibiting the expression of the E2F1 gene in thenucleus, E2F1 gene expression being a sign of cell proliferation. Thus,a decrease in the association of LIV21 with the E2F4 protein isindicative of the presence of cancer cells. Similarly, the presence ofthe E2F1 protein in the nucleus is indicative of the presence of cancercells.

Consequently, the present invention concerns a method for the detectionof cancer cells in a biological tissue sample (for example, breast,ovary, endometrium, bladder, melanoma, prostate, glioblastoma, etc.)from patients, this method comprising the detection of the products ofexpression of the LIV21 complex genes in the nucleus comparatively tothe same products in the cytoplasm and the membranes of the cells in thebiological tissue sample from said patient, this method comprisingdetection of the product of expression of liv21F gene in the core and/orthe cells cytoplasm in the biological tissue sample from said patient, alocalization of said products of expression of the LIV21F gene in thecytoplasm is indicative of the presence of cancer cells and alocalization of said products of expression of the LIV21 complex genesin the nucleus is indicative of the presence of noncancer cells.Preferably, a localization of said products of expression of the LIV21gene in the cytoplasm is indicative of the presence of invasive and/ormetastatic cancer cells, the localizations of the products of expressionof the LIV21 complex genes and its associated partners described in theexamples of biochips shows or not the cancer cells presence. Optionally,the method according to the present invention also comprises thedetection of the product of expression of at least one gene selectedfrom the group consisting of the protein kinase C epsilon (PKCs) gene,the E2F1 gene and the E2F4 gene. The method can in particular comprisethe detection of the product of expression of two of these genes or ofthe three genes. Moreover, at least one of the ratios LIV21/PKCS,LIV21/E2F4 and LIV21/E2F1 can be determined in the present method. Thisratio can be determined in the cytoplasm and/or in the nucleuspreferably separated. Preferably, these ratios are determined in thenucleus. Preferably, these ratios are compared with those obtained in anormal cell. The level of expression of each enzyme or polypeptide ofthe SUMO/Rb/HDAC complex or, for certain cell types, of thePML/SUMO/Rb/HDAC complex is an additional indicator of the proliferativestate of the cell.

These ratios of expression or silencing can be detected via the proteinexpression or inhibition level themselves in the protein arrays(biochips) fabricated according to conventional methods described(Lubman David M, QIAO TIECHENG Alex, Mathew ABY J etc.) or novel toolsfor the automation of hybridization and of reading, US2004152212 and YuXinglong US 2005019828 and novel supports which attach polypeptides(patents US 2008 213130 and US 2005/0157445 or US 2006170925 or WO 2005016515, Klages Claus Peter and example figure).

Before describing the principle of these biochips, which are well knownby man skilled in the art, we will give the following definitions: Thebiological sample can be in particular sample of blood, serum, saliva,tissue, tumor, bone marrow, circling cells from the patient. Thebiological sample can be recovered by any type of sampling know by thoseskilled in the art. According to the present invention, we consider abiological sample any material allowing the detection of expression of atarget gene. The biological material can include in particular proteins,or nucleic acids such as desoxyribonucleoic acid (DNA) and rybonucleicacid (RNA). The nucleic acid can in particular be an RNA (rybonucleicacid). According to a preferred embodiment of the invention, thebiological material comprises nucleic acids, in particular RNAs and evenmore specifically total RNAs. Total RNAs include transfer RNAs,messenger RNAs (mRNAs), such as mRNAs transcribed from the target gene,but also transcribed from any other gene, and ribosomal RNAs. Thisbiological material includes specific material of the target gene, suchas in particular mRNAs transcribed from the target gene or proteinsissued from these RNAs, but it can also include material unspecific tothe target gene, such as in particular mRNAs transcribed from a genedifferent from the target gene, tRNAs, rRNAs issued from other genesthan the target gene. When the extracts to be studied consist of cellcultures, they will preferably be analyzed on fresh cultures (with orwithout previous treatment) that underwent an extraction protocolseparating cellular compartments. This type of kit known by thoseskilled in the art allows the specific extraction of membranes or solelythe cytoplasmic or nuclear or cytoskeletal content in a differentialfashion by using different solutions. For instance, the kit Proteoextract (ref 539790) from Calbiochem can be used.

The other aspect of the present invention is the use of the genes andthe proteins mentioned above as markers for the invasiveness and themetastatic aggressiveness of cancer cells of the prostate,—colon,bladder, melanoma, ovary, endometrium and cervix, and cancers inneurobiology or in ORL etc. . . . In fact, sequential pharmacodiagnostictests during treatment monitoring will permit to observe, by comparingat different time points, variations of the expression level or of theirsilencing and therefore to better evaluate the treatment efficiency, toreadjust these treatments in the case of a suitable multitherapy in sucha way that the physiological equilibrium of the different products ofgenes involved in metabolic complexes are maintained. The plasticity ofthese equilibriums justifies the use of diagnostic biochips and for thetherapeutic monitoring with the most pertinent genes of metaboliccomplexes involved in the physiology of anarchic proliferation in thecase of breast cancer, which is highly heterogeneous. Therefore eachindividual or each phenotypic subgroup of individuals will show an underor over expression profile of genes of the metabolic complexes which isspecific to him.

In one embodiment, the expression product of the genes is detected atthe mRNA level. mRNA can be detected by RT-PCR analysis (i.e. followingexamples). It can also be detected by Northern blotting analysis or bySPR if the RNAm and DNA are functionalized at the surface ofelectroapplied on a support of biochip (techniques described thereforein the patents US 2008 213130 and US 2005 0157445 or US 2006 170925 orWO 2005 016515). The MICAM technique which uses the electric piezoeffect in its biochips can be also used for the above mentionedinvention of pharmaco diagnostic biochip dedicated to the cerebralneuroblastoma and other cerebral cancers, of biochips diagnosticdedicated to the epidermoid cancers and more specifically dedicated tothe breast, ovarian and prostate cancers.

In an alternative mode of realization, the product of gene expression isdetected on the level of protein or peptides characterizing complexLiv21 and its partners of interaction. Preferably, the protein and/orproteinic complex Liv21 associated, are detected using an specificantibody. For example, the protein can be detected by analysis WesternBlot and SPR, a system of biochip using a wave of transverse propagation(evanescent wave) of surface plasmonic resonance (SPR). The interactioncan be done with an electronic surface, conducting semi surface createsan exiton by luminescence or fluorescence. In a mode of preferredembodiment, it is detected by immuno histochimy, immuno cytochemistry,microfluidic, radiography or peroxidase labeling or any other means ofoptical, sonic imagery or of spectroscopy.

In one specific embodiment of the method comprising the detection of theexpression product of the PKCε gene, a significant increase in PKCε isindicative of the presence of cancer cells. Moreover, the method canalso comprise the determination of the LIV21/PKCε ratio in 5 thenucleus, the membranes and the cytoplasm. This ratio can be comparedwith the one observed in a normal cell. An increase in the LIV21/PKCεratio in the cytoplasmic fraction is indicative of cancer cells.

In another specific embodiment of the method comprising the detection ofthe expression product of the E2F4 gene, the method comprises thedetection of the association of LIV21 with the E2F4 protein, a decreasein this association in the cell nucleus being indicative of the presenceof cancer cells. Moreover, the method can also comprise thedetermination of the LIV21/E2F4 ratio in the nucleus and/or thecytoplasm. This ratio can be compared with the one observed in a normalcell.

In an additional embodiment of the method comprising the detection ofthe expression product of the E2F1 gene, the presence of the E2F1protein in the nucleus is indicative of the presence of cancer cells.Moreover, the method can also comprise the determination of theLIV21/E2F1 ratio in the nucleus and/or the cytoplasm. This ratio can becompared with that observed in a normal cell.

In a specific embodiment, the method comprises the detection of alabelled small interfering RNA (siRNA) in order to target its specificsequence and therefore signal the locus of messenger RNA expression ofthe gene of interest. In this way, the specific small interfering RNAcan be used as a diagnosis marker similarly to an antibody. The specificsiRNA would allow to locate in a specific case such as in extamporaneoustissues or any kind of sample from a patient, such as cancer tissuessample, the fluorescence signal or any other marker used on the siRNA isfound in a cellular compartment on the sample. An siRNA targeting theE2F1, E2F4 and PKC epsilon would allow a complementary diagnosis.

The method according to the present invention allows in particular thedetection of metastasized cancer, therapeutic monitoring and/orrecurrences following treatment.

A second aspect of the invention concerns the human LIV21 protein andalso the fragments thereof. More particularly, the present inventionconcerns a purified or recombinant isolated human LIV21 protein. Itconcerns in particular an isolated polypeptide comprising a peptidesequence selected among SEQ ID Nos 1 to 5 and more broadly selectedamong the peptide sequences characterizing it and obtained by MALDI(FIGS. 3, 4 and 5) and NanoLC-ESI-MS. In a preferred embodiment, thepolypeptide comprises the three peptide sequences SEQ ID Nos 1 and 2 and3. Preferably, the LIV21F protein and certain proteins of the Liv21complex comprises a leucine zipper motif, a basic domain characteristicof DNA binding domains (FIG. 2), and a nuclearization sequence.

In an even more preferred embodiment, the present invention concerns thepolypeptides with peptide sequences characterized by spectrograms ofFIGS. 3, 4, 5 of gel bands 1, 2 and 3, selected among SEQ ID Nos 1 and 2and 3 and 4 and 5 and a hundred additional non ordered sequencessupplement (i.e. listing sequences in annex), the other sequences of theproteins must being checked compared with their homologies withcontaminants and order during spectrometries of mass MSMS on theunmatched fragments identified in the Maldi T of analysis (i.e. FIGS. 3,4, 5), these unmatched fragments corresponding to the masses M (H+)untagged characterized in part the Liv21 protein and some elements ofthe Liv21 protein complex.

A third aspect of the invention concerns an antibody, which the presentinvention. More particularly, the antibody can bind specifically to apolypeptide comprising a peptide sequence selected from SEQ ID Nos1-180, preferably from SEQ ID Nos 1 and 2 or 3 and/or 5 or 51, or asequence having more than 80% identity to said sequences. The presentinvention concerns in particular an anti-LIV21 serum produced byimmunizing an animal or a human with a polypeptide according to thepresent invention, in particular a polypeptide comprising a peptidesequence selected from SEQ ID Nos 1-180, preferably from SEQ ID Nos 1-5and 51, or a sequence having 70%, 80% or 90% identity to said sequences.

A fourth aspect of the invention concerns a kit for the detection ofcancer cells in a biological sample from a patient, this kit comprisingone or more elements selected from the group consisting of an antibodywhich binds specifically to human LIV21F according to the presentinvention and an anti-LIV21F serum according to the present invention, aspecific oligonucleotide mRNA probe of Liv21F and a pair of primersspecific of mRNA. In a specific embodiment of the invention, the kitalso comprises means for detecting the product of expression of a geneor a specific oligonucleotide mRNA probe of factors selected from thegroup consisting of the protein kinase C epsilon (PKCc) gene, the E2F1gene and the E2F4 gene. But also the antibodies in a specific antibodiesmicroarray from the antibodies group consisting of RBP2, SUMO, HDAC,TNFalpha, crb2, cycE/cdk2, cdk1, CREB1, p300, Rb, p107, p130, NFkB,cdc2A, mdm2, p21, p53, p65. It also comprises Microarray with saidproteins above and specific peptides known by a person skilled in theart, their antigens being referenced. The combination of these differentpeptides corresponding to the specific interactions of protein complexesacting in metabolic deregulation, induces anarchical proliferation,which is a specific feature of cancer or neurodegeneration. Theinvention concerns the use of an antibody specific for human LIV21 forthe diagnosis of cancer, and antibodies specific for its proteincomplex, but also specific antibodies for RBP2, SUMO, HDAC, TNFalpha,crb2, cycE/cdk2, cdk1, CREB 1 and p300, Rb, p107, p130, NFKB, cdc2A,mdm2, p21, p53, p65, p73. Also, the invention concerns the used ofprimers pair or LIV21 specific probe for the cancer diagnosis.Preferably, the diagnosis is performed ex vivo on samples from a patient(puncture of the cerebral spinal fluid, blood test, biopsy, groundcellular material, bronchial aspirations, DNA/protein/antibodies arrays,plasmionics (SPR), hydrophobic or ion metal supports, etc). Methodaccording to claim 15, characterized in that in addition, it implementsat least any of the specific probes of the sequences of known as saidcomplex Liv21 and these associated partners. The method is characterizedin that the aforementioned biochip is: a biochip with protein, or abiochip with nucleotidic antibodies or a biochip with acids, or abiochip with mRNA, or a biochip with SiRNA.

Method is characterized in that the aforementioned biochip with protein,or the aforementioned biochip with antibody, consists of a biochip offluidic microcomputer and for which the aforementioned stage ofdetection consists of a detection by SPR.

Method is characterized in that it understands a stage ofamplification/retro-transcription by RT-PCR of at least a nucleotidicsequence of the known as human Liv21 complex according to claim 1 or 2or of the known as Liv21 complex and its associated partners.

Method is characterized in that it understands a pharmaco-diagnostictest for diagnosis of the cancer or the follow-up of the development ofa cellular proliferation, the aforementioned cancer being preferentiallychoose in the group consisted the neuroblastome, the glioblastomas andother cancers touching tissues of the nervous system.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1A: one-dimensional gel (acrylamide gradient 12%) revealing afterthree hours thirty of migration 11 tapes with certain triplets anddoubled tapes and a two-dimensional gel SDS Page of total cellularextracts with spots between 15 and 20 KD and to 29-32 kD and 35 KDapproximately with basic pH and of the spots with 190-180 and to 100 kDwith acid pH;

FIG. 1B: diagram of the interactions of the bio-markers of complex Liv21and the surrounding metabolic pathways;

FIG. 2: scheme of nuclear and cytoplasmic protein with domain DNAbinding and effects on the study of therapeutic targeting the core andthe cell cytoplasm;

FIG. 3A: The listing of monoisotopic peaks of the band 1 at 50 kD andthe band 2 between 49 and 50 kD;

FIG. 3B: LIV21 protein profile by mass spectrometry (Maldi) M (H⁺) forthe one-dimensional gel band corresponding to the band 2 migrating at49-50 kD wherein the peptides derived from the digestion are solubilizedin a solvent: acetonitrile/water (1/1) containing 0.1% of TFA(trifluoroacetic acid);

FIG. 4A is a profile of the spectrogram of the band 6 named 6FC;

FIG. 4B: monoisotopic of certain peaks from FIG. 4A;

FIG. 5 is the third spectrogram corresponding to the one-dimensional 12%acrylamide gel band migrating at 52 kD and revealed with coomassie blueand the LIV21 antibody;

FIG. 6: analysis on the data banks the listings of monoisotopic peaks;

FIGS. 7A and 7B relate to SEQ ID NO 217;

FIG. 8: RNA pool;

FIG. 9: PCR with housekeeping genes and analysis of molecular masses;

FIG. 10: PCR with the primers showing a band of 1400 bp;

FIGS. 11A & 11B: Gel 2 with analysis of molecular masses;

FIG. 12: Gel 3 at 55° and analysis of molecular masses;

FIG. 13: Gel 4 at 45° and at 55° and analysis of molecular masses;

FIG. 14: screening ligation of 400 pb band, clones B1 to B10;

FIG. 15: screening ligation of 1400 pb band, clones CI to C10;

FIG. 16: Gel 5: ligation screening on the five new clones;

FIG. 17: Gel 6: Screening of the S55T and S55M recombinant clones andanalysis of molecular masses;

FIG. 18: examples of comparison of nucleotide sequences between thesequenced clones;

FIGS. 19A & 19B: Si RNA design;

FIGS. 20A-20D: Protein biochip (array) from Yeretssian but in additionwith peptides named of the proteins of the interested complex studied inthe invention;

FIG. 21: Two biochips standard microfluidic of four shafts x2 with acontrol and three biomarkers;

FIG. 22: Example of biochip of 20 spots with 16 biomarkers of interest(and four controls) fixed on the sensorchip allowing to see by SPRon-expression and the under-expression of certain genes of complex LIV21and its partners of interactions for example;

FIGS. 23A & 23B: Biochip with RNA allowing to explore mini RNA ofcomplex LIV21; and

FIG. 24: Example of biochip with DNA resulting from above mentionedgenes of interest targeting pathology.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1A: one-dimensional gel (acrylamide gradient 12%) revealing afterthree hours thirty of migration 11 tapes with certain triplets anddoubled tapes and a two-dimensional gel SDS Page of total cellularextracts with spots between 15 and 20 KD and to 29-32 kD and 35 KDapproximately with basic pH and of the spots with 190-180 and to 100 kDwith acid pH. Spots between 49 and 51 KD and to 64 KD. Intomono-dimensional: Tapes with ISO kD approximately (TOFC), 100 KD, 64 kD,51 à49 kD, 49 kD, 35 kD, 29 kD, 15 to 17 kD.

FIG. 1B: diagram of the interactions of the bio-markers of complex Liv21and the surrounding metabolic pathways.

FIG. 2: scheme of nuclear and cytoplasmic protein with domain DNAbinding and effects on the study of therapeutic targeting the core andthe cell cytoplasm.

FIG. 3 A: The listing of monoisotopic peaks of the band 1 at 50 kD andthe band 2 between 49 and 50 kD. FIG. 3B: LIV21 protein profile by massspectrometry (Maldi) M (H⁺) for the one-dimensional gel bandcorresponding to the band 2 migrating at 49-50 kD. The peptides derivedfrom the digestion are solubilized in a solvent: acetonitrile/water(1/1) containing 0.1% of TFA (trifluoroacetic acid). A saturatedsolution of the alpha-cyano-4-hydroxycinnamic acid matrix is prepared inthe same solvent. The same volume of the two solutions is taken andmixed together, and 1 microliter is deposited onto the Maldi plate foranalysis.

FIG. 4 is a profile of the spectrogram of the band 6 named 6FC.

The de novo analysis (MS MS Maldi T of T of) makes it possible topropose the sequences: RYLVTPVNA, RYVPSSNLP, RYVLSPVK, RYVPSSNPL,RYLPSANPD.

FIG. 4 bis: monoisotopic of the peak 1032.58 MSMS analyzes: YRPGTVALR,RYVPSSNPL

The peak monoisotopic 944.6 is a sequence: FAVAFPVGR

The peak monoisotopic 1603.7: KPSHPKPSTK

The peak 1328.69: AHNLFKT, TFKNLC

The common peaks monoisotopic between the first 2004 (230304 imagenium03_H11_a_(—)001 and the peak 6FC from 2008 are:

Following peaks monoisotopic: 1135.563, 1151.545; 1167.604; 1206.589;1324.634; 1336.658; 1507.735; 1604.710; 1800.940; 2087.034.

The variation of the protocol is due only to three different stages: thefirst is the purification of the antibody used, the second is theseparation of the fractions cytoplasmic and nuclear, the third is theheating two minutes with 100° of the sample before migration on freezingof acrylamide.

FIG. 5 is the third spectrogram corresponding to the one-dimensional 12%acrylamide gel band migrating at 52 kD and revealed with coomassie blueand the LIV21 antibody.

FIG. 5 bis is a table of the monoisotopic peaks of the third spectrogramcorresponding to the one-dimensional acrylamide gel band migrating at 51kD 52 kD and revealed with coomassie blue and the LIV21 antibody.

The monoisotopic peaks with a value M H+. The masses are give with threenumbers after the decimal point by the proteomic platforms since theyestimate that this is the acquisition precision limit of MALDI TOFmachines. The FIGS. 3-5 describe the MALDI analyses giving a set ofpolypeptides that can be assigned to the LIV21 protein and its complexand contaminants sometimes different according to the observers from thevarious platforms of proteomics under discussing.

FIG. 6: analysis on the data banks the listings of monoisotopic peaks.Example of the histatine 3. The Mascot search parameters are: trypsinenzyme, variable modifications: carbamethylation and oxidation ofmethionins, without molecular mass limit, without isoelectric pointrestriction.

FIG. 7 idem but the second example:

Type of mass: monoisotopic. Mass error (MS): according to the observer50 ppm or 100 ppm. Non-cleavage with trypsin: 1 The masses captured areM (H⁺)/real masses. For spectrogram 1, the cysteins are blocked withiodoacetamide. The possibility of digestion with Promega bovine trypsinmay be incomplete with cleavage oversight.

Sequences common with Gallus gallus (gi 50732569), the 30 MouseSyntaxin, the histatin variant HIS3-2 (P15516-00-01-00), theZN575-Human, the G6P translocase, the HSP60 chaperonin, the deiminase,ferrodoxin NADP(+) reductase, pseudomonas polyribonucleotidenucleotidyltransferase, the clathrin, the dehydrolipoamidedehydrogenase.

FIG. 8: RNA pool

FIG. 9: PCR with housekeeping genes and analysis of molecular masses.

FIG. 10: PCR with the primers showing a band of 1400 bp.

FIG. 11: Gel 2 with analysis of molecular masses FIG. 12: Gel 3 at 55°and analysis of molecular masses

FIG. 13: Gel 4 at 45° and at 55° and analysis of molecular masses.

FIG. 14: screening ligation of 400 pb band, clones B1 to B10.

FIG. 15: screening ligation of 1400 pb band, clones CI to C10.

FIG. 16: Gel 5: ligation screening on the five new clones. FIG. 17: Gel6: Screening of the S55T and S55M recombinant clones and analysis ofmolecular masses.

FIG. 18: examples of comparison of nucleotide sequences between thesequenced clones.

FIG. 19: Si RNA design.

FIG. 20: Protein biochip (array) from Yeretssian but in addition withpeptides named of the proteins of the interested complex studied in theinvention.

FIG. 21: Two biochips standard microfluidic of four shafts x2 with acontrol and three biomarkers.

An on-expression of DDX1, MYCN and CRABPII is observed whereas oneobserves by testing the second sensorship an under-expression of HUD,TP53 INP1 and a major under-expression of p21.

FIG. 22: Example of biochip of 20 spots with 16 biomarkers of interest(and four controls) fixed on the sensorchip allowing to see by SPRon-expression and the under-expression of certain genes of complex LIV21and its partners of interactions for example.

An under-expression of DKK1, SKP2, DKK3, ID2, p21, SKP2, TP531NP1, ID2,P73 is observed whereas an on-expression of MYC N, ALK, NLRR1 (theleucine rich neuronal repeat is transactivated), CRABPII, DDX1, LIV21K,AURORA kinase A is observed.

FIG. 23 Biochip with RNA allowing to explore mini RNA of complex LIV21and more particularly those resulting from genes of the polypeptidesLIV21F and LIV21K added in Mir RNA, microRNA known to be implied inpathology ((miR (=MIR) 34A. MIR9. MIR125A.125B.MIR128 MIR184 MIR221)).

We study also regulator PTEN and factors

TR AIL.

FIG. 24: Example of biochip with DNA resulting from above mentionedgenes of interest targeting pathology.

The invention relates to the identification of antigens in cell lysatesby immunoprecipitation. The analysis of the physical interaction ofvarious proteins associated in the LIV21 complex as E2F4 and E2F1 hasbeen studied by coimmunoprecipitation of protein complexes. Thisanalysis has made it possible to demonstrate novel 10 markers, which hasa diagnostic and prognostic use for cancer (i.e. PCT/FR2006/000510).Begin by using the one dimensional and two dimensional gelelectrophoresis analysis (FIGS. 1 and 2), the protein samplescorresponding to the putative protein and to the elements of the complexwere extracted from the gels and digested with trypsin (Promega) inorder to be analysed by MALDI (FIGS. 3 to 5) and ESI MS/MS massspectrometry. The results, when put up against proteomic databanks, madeit possible to reveal several peptide sequences of interest, includingsome given as an example (FIGS. 6 and 7), some being found in humanswith very significant scores (splicing of histatin, etc., FIGS. 8 and9), these sequences were used as primers (once reverse-transcribed tocDNA) for screening a library formed from breast cancer-specific MCF7cells (FIGS. 10 to 17).

The cloning made it possible to bring to the fore about twenty clonesout of the 150 clones obtained, of which ten clones were sequenced andcharacterize the new LIV21 gene LIV21F and the gene LIV21K (FIG. 1B andFIG. 2). Based on these sequences, siRNAs were determined in order toallow regulation of silencing type within this metabolic complex ofinterest so as to develop therapeutic applications (FIG. 18).

In post-mitotic cells, apoptosis could correspond to an aborted attemptat mitosis. It is in this context that the application of LIV21 has beendeveloped. The inventor has identified sequences of the LIV21 gene.Using the LIV21 antibody on affinity columns he has been able to extractpeptides of the LIV21 protein; it has also used a second approach bymeans of a coimmunoprecipitation kit (Pierce) in order to have largeramounts of proteins (Example 1). Based on peptide sequences of the LIV21protein, obtained by mass spectrometry (Example 2), primers which makeit possible to amplify a cDNA fragment were designed (Example 3). Afterculturing and amplification of MCF7 line cells, extraction andpurification of RNAs, RT PCRs and cloning in a shuttle vector werecarried out, and then screening of the resistant colonies and sequencingmade it possible to reveal sequences characterizing the genes LIV21F andLIV21K (Examples 4 and 5). More than twenty characteristic clones out of150 clones were studied. The cDNA of these clones was used to screen alibrary prepared from the total mRNA of MCF7 cells.

The sequence of this new products are new transcription factors, thenuclear translocation of which change their role and their function,that being correlated for some of them with the establishment of thecellular quiescence such as for example LIV21F, E2F4. In addition it islinked to the DNA role and function. Furthermore, it forms heterodimerswith other transcription factors and certain bind to DNA.

Using Northern blotting, the inventor then followed the expression ofthis new product during development, from the embryonic stage. It wasobserved that the amount of the LIV21 protein increases as developmentprogresses, i.e. as a quiescent cell state becomes established. Throughthe same strategy, the inventor showed that the LIV21/E2F4 complexinhibited the expression of E2F1. This complex could correspond to a newcheckpoint in the arrest of cell proliferation.

LIV21 complex and associated metabolic complex:

The present invention relates to the LIV21 complex and its newnucleotide sequences SEQ ID No. 171 to SEQ ID No 217 also used for someof them in the form of siRNA for diagnostic and therapeutic applicationsand also LIV21F and LIV21K polypeptides and derivatives and fragmentsand isoforms thereof (SEQ ID No to SEQ ID No 215).

LIV21 human complex, characterized in that it comprises:

the nucleotide sequences SEQ ID No 171 to 175, andsiRNA sequence derived from one of the RNA sequences SEQ ID No 120 andSEQ ID No 121, and 171 to 175a protein fraction comprising at least sequence SEQ ID No 1 and 181 or asequence having 90%, and preferably 80%, and more preferably 70%identity with said SEQ ID No 1 and 181.

LIV21 human complex, characterized in that it comprises:

The nucleotide sequences SEQ-ID No 171, 172, and the protein fractionscomprising at least sequence SEQ ID No 1 or a sequence having 181 or 70,10 80 or 90% identity with said SEQ ID NO 1 or 181 and SEQ ID No 183 ora sequence having 70, 80 or 90% identity with said SEQ ID No 183.

LIV21 complex human, characterized in that it also comprises:

nucleotide sequences SEQ ID No 123, 124 and 127, and the proteinfractions comprising at least sequence SEQ ID No. 1 or a sequence having70, 80 or 90% identity with said SEQ ID NO 1 and the sequence from 181to 185 or a sequence having 70, 80 or 90% identity with said SEQ ID NO181 to 185.

LIV21 complex human characterized in that it further comprises:

Any of the nucleotide sequences or ribonucleic SEQ ID No 119, 120, 121,122, 123, 124, 125, 126 or 127 or a sequence having 90%, and preferably80%, and more preferably 70% identity with said sequence SEQ ID No 119to 127, or

UUGGUAACGACCAUGCCAC, or UUCACUUAGAAUAAUGUCC, or UCUUUGUGAAUUUGACAAC, orUCAAGGUCCAGGCUACAAC, or

Any of the siRNA following:

GUGGCAUGGUCGUUACCAA dTdT dTdT CACCGUACCAGCAAUGGUUGGACAUUAUUCUAAGUGAA dTdT dTdT CCUGUAAUAAGAUUCACUUGGAAGAAUCUCAUCUCAGAUUCAA ) UUCCUUCUUAGAGUAGAGUCUAGAG-GCAGAUCAUGAGGUCAAGAUUCAA ) UUCGUCUAGUACUCCAGUUCUAGAG-GAAGAAUCUCAUCUCAGAAUUCAA ) UUCUUCUUAGAGUAGAGUCUUAGAG-GUGUGAGACUCCAUCUGAAUUCAA ) UUCACACUCUGAGGÜAGACUÜAGAG-GAUCAUGAGGUCAAGAGAUUUCAA ) UUCUAGUACUCCAGUUCUCUAAGAG-GAGAGUCAÜCUUACUCAGAUUCAA ) UUCUCUCAGUAGAAUGAGUCUAGAG-GCUGGGUGUGGUAGUGCAUUUCAA ) UUCGACCCACACCAUCACGUAAGAG-GUCAAGAGAUCGAGACCAUUUCAA ) UUCAGUUCUCUAGCUCUGGUAAGAG-GUCAUCUUACUCAGAGCAUUUCAA ) UUCAGUAGAAUGAGUCUCGUAAGAG-GGCUGAGGCAGGCAGAUCAUUCAA ) UUCCGACUCCGUCCGUCUAGUAGAG-GGAGUAUAGGAAUCUCCUAUUCAA ) UUCCUCAUAUCCUUAGAGGAUAGAG-GGUAGUGCAUGCCUGUAGUUUCAA ) UUCCAUCACGUACGGACAUCAAGAG-GAGAUGGCGCCACUGUACUUUCAA ) UUCUCUACCGCGGUGACAUGAAGAG-GCCUGGCGACAGUGUGAGAUÜCAA ) UUCGGACCGCUGUCACACUCUAGAG-GCCUGUAGUCCCAGCUACUUUCAA ) UUCGGACAUCAGGGUCGAUGAAGAG

LIV21 complex and associated metabolic complex:

The present invention relates to the LIV21 complex and its newnucleotide sequences SEQ ID No. 171 to SEQ ID No 217 also used for someof them in the form of siRNA for diagnostic and therapeutic applicationsand also LIV21F and LIV21K polypeptides and derivatives and fragmentsand isoforms thereof (SEQ ID No to SEQ ID No 215).

LIV21 human complex, characterized in that it comprises:

the nucleotide sequences SEQ ID No 171 to 175, andsiRNA sequence derived from one of the RNA sequences SEQ ID No 120 andSEQ ID No 121, and 171 to 175a protein fraction comprising at least sequence SEQ ID No 1 and 181 or asequence having 90%, and preferably 80%, and more preferably 70%identity with said SEQ ID No 1 and 181.

LIV21 human complex, characterized in that it comprises:

The nucleotide sequences SEQ-ID No 171, 172, and the protein fractionscomprising at least sequence SEQ ID No 1 or a sequence having 181 or 70,10 80 or 90% identity with said SEQ ID NO 1 or 181 and SEQ ID No 183 ora sequence having 70, 80 or 90% identity with said SEQ ID No 183.

LIV21 complex human, characterized in that it also comprises:

nucleotide sequences SEQ ID No 123, 124 and 127, and the proteinfractions comprising at least sequence SEQ ID No. 1 or a sequence having70, 80 or 90% identity with said SEQ ID NO 1 and the sequence from 181to 185 or a sequence having 70, 80 or 90% identity with said SEQ ID NO181 to 185.

LIV21 complex human characterized in that it further comprises:

Any of the nucleotide sequences or ribonucleic SEQ ID No 119, 120, 121,122, 123, 124, 125, 126 or 127 or a sequence having 90%, and preferably80%, and more preferably 70% identity with said sequence SEQ ID No 119to 127, or

UUGGUAACGACCAUGCCAC, or UUCACUUAGAAUAAUGUCC, or UCUUUGUGAAUUUGACAAC, orUCAAGGUCCAGGCUACAAC, or

Any of the siRNA following:

GUGGCAUGGUCGUUACCAA dTdT dTdT CACCGUACCAGCAAUGGUUGGACAUUAUUCUAAGUGAA dTdT dTdT CCUGUAAUAAGAUUCACUU

LIV21 complex human (figures let 2) characterized in that it furthercomprises at least:

-   -   Any one of the nucleotide sequences SEQ ID No 123, SEQ ID No 124        and SEQ ID No 127 to 149, or SEQ ID No 217    -   any one of amino acid sequences SEQ ID No 1 to 148 and SEQ ID No        150 to 170 and SEQ ID NO 180 to 185, or a sequence having 90%,        and preferably 80%, and more preferably 70% identity with said        sequence SEQ ID No 180 to 185 and SEQ ID 1,2,5 and SEQ ID No 215

LIV21 Complex human characterized in that said protein fraction furthercomprises any of the following proteins: E2F1, E2F4, p130, p300, p107,Liv21F, HDAC-1, PML, SUMO et PKC epsilon, Aurora A, Survivin, BCAS4,BCAS3, RFSH.

LIV21 complex human characterized in that it interacts with at least oneof its associated partners, at least one of its associated partners areselected from the group consisting of: any one of the followingproteins: RBP2, TNFalpha, crb2, cycE/cdk2, cdk1, CREB1, p300, p107,NFkB, cdc2A, mdm2, p21, p53, p65, p73 MYC, NMYC, TGFbéta, Chlatrin,Aurora, AKT, BRCA1, F0X04 or cyclin A et D1, CHUK, HMGA2, IKBKB

-   -   an antibody of anyone of the following proteins: RBP2, E2F4,        E2F1, SUMO, HDAC-1, crb2, Int2, cmd2, cycE/cdk2, cdk1, CREB1 et        p300, Rb, p 107, p 130 of the family of pocket proteins NFkB,        cdc2A, mdm2, p21, p53, p65, p73, the cyclin A and D1, CHUK, MYC,        NMYC, TGF Beta, Chlatrin (2), Aurora, AKT, BRCA1, F0X04, HMGA2,        BCAS3, BCAS4, solute carrier.    -   in that it comprises an extract of proteins and peptides        obtained by latching to a polyclonal antibody of the LIV21, and    -   in that its electrophoretic profile acrylamide gel comprises at        least three bands: band of 50 kD, 51 kD band, and the band of 52        kD.

Complex human LIV21 is also characterized in that, after trypsindigestion, the profile of MALDI includes at least mono isotopic peaksfollowing:

1135.563; 1151.545; 1167.604; 1206.589; 1324.634; 1336.658; 1507.735;1604.710; 1800.940; 2087.034.

Method of detecting the complex human LIV21, characterized in that itcomprises the implementation of at least one probe specific for at leastone sequence of said complex LIV21 according to claim 1 or 2.

Method of detecting the complex human LIV21, characterized in that itfurther comprises the implementation of at least one probe specific forat least one sequence of the LIV21 said complex and its associatedpartners.

Method of detection of the complex and its partners LIV21 in that it iscomprises at least the steps of:

-   -   A step of extracting biological material from a biological        sample taken from a patient,    -   A step of contacting said biological material with at least said        probe specific for any of the sequences of said human or complex        LIV21 or LIV21 said complex and its content partners, and at        least one control and    -   A step of detecting the expression of the expression products of        the genes of said complex of said human or LIV21 LIV21 complex        and its content partners, said products being comprised of        expression of messenger RNA, or peptides, or proteins.

Detection method is characterized in that it also aims to screen acandidate compound, said candidate compound is capable of modulating theactivity of the complex human LIV21, and that it also includes:

a step of contacting said biological material with said candidatecompound, and a step of selecting said candidate compound.

Method is characterized in that said biological sample is taken from acancer patient and in a healthy patient, and in that said biologicalmaterial comprises cell nuclear extracts, and cytoplasmic cell extracts,cell membrane extracts, and in that it further comprises a step ofdetermining of the under-expression and overexpression of the geneproducts of said complex of said human or LIV21 LIV21 human complex andits content partners said biological extracts.

Method is characterized in that it further comprises a step ofdetermining the ratios of said sub-expression and overexpression of saidgene products: cell nuclear extracts, and cytoplasmic cell extracts,cell extracts or membrane, and in that it further comprises a step ofsuch analysis combined ratios of said biological material taken from acancer patient and in a healthy patient.

Method is characterized in that said probe comprises a sequence specificsiRNA labeled with rhodamine.

A method according to any one of the above approaches is, characterizedin that it implements a biochip, on which is deposited at least onesequence-specific probes from said complex human LIV21.

Method is characterized in that, in addition, it implements at least onespecific probe sequences LIV21 said complex human and associatedpartners

Method is characterized in that said biochip is a protein biochip, or abiochip antibody, or a nucleotide acid microarray, or a RNAm biochip, ora siRNA biochip.

Method is characterized in that said detecting step implements any meansof optical imaging, or any means of sonic or any means of spectroscopy.

FIG. 1 characterizing the protein complex, and its PI and its PM aredependent on the temperature and conditions of migration and itsobservation in total extracts or in extracts of cellular compartments.

FIGS. 1, 2, 3). It gives more than 54 peptides following digestion withPromega trypsin (FIG. 7). The characteristics of the LIV21F protein arealso described in FIGS. 1, 2, 3, 4. Twenty specific peptides of LIV21have been described: LIV21a (SEQ ID No 1) and LIV21b (SEQ ID No 2) atthe sequence 180.

The gene of this protein is characterized by two main sequences (i.e.patent in listing) and sequences representing an alternate splicing.

LIV21K corresponds to a sequence of strong homology (of more than 60%)with AD7cNTP, the neural thread protein in N terminal (position 193 to299) of (060448_Human). SEQ NoSVAQAGVQWRNLGSLQALPPGFMPFSCLSLLSSWDYRRLPPRPATFLYFPRQGFTVLARMVSISPRDPPASASQSVGIAYISNFFFFEMESRSVAQAGVQWHNLGSLQALPPGFMPFSCLSLLSSWDYRRLPPRPATFLYFPR

With variable ones within peptide in position (i.e. figure . . . ):

The structure of LIV21K is like that of the AD7C NTP, i.e. repeatedreasons that can be also analyzed under reasons which correspond tofunctional fields.

SVXQAGVQWXNLGSLQXLPPGXXXFSCLSLXSSWDYXXLPPXPAXF

Or a variable one:

SVXQAGVQWXNLGSLQXLPPGFXXFSCXSLSSWDYRRXPPRXA

Between these two repeated reasons, there is a peptide of 40 amino-acidswith a reason also preserved:

ISPXDXPASASQSXGIXXXSX THE LIV21I: FLYFPRQGFTVLARMVSISPRDPPASASQSVGIAYISN

With alternatives in position:

For example for LIV21I

In position 31, an A instead of the V and in position 34 a T instead ofthe amino-acid A. Thus: the amendment of the end PASASQSAGIT but also analternative where the A becomes T in position 25: . . . DPPTSASQSVGIIdem for LIV21F or K:

SEQ ID No: FSCLSL L SSWDYRR L PPRPA T FLYF where the amino-acid inposition 7 here becomes a proline (P instead of L) and/or in position 15too (with the possibility of an alternative Alanine; sometimes theposition 14 changes of a R in H and/or in position 21 where anamino-acid T becomes N.

SEQ ID No: FSCLSL P SSWDYRR P PPRPA N FLYF

Moreover, two other peptides of LIV21 are also described: the peptideLIV21c (SEQ ID No 3) and the peptide LIV21d (SEQ ID No 4). The peptideLIV21e (SEQ ID No 5). KFFVFALILALMLSMCGADSHAKR with which the finalsection is homologous with a sequence with LIV21K.

Other specific peptides of LIV21 are described in the additional list.

A homology with a functional segment of the protein zinc finger 575(ZN575-Human): score 13 to 48 and 91% of recovery

A homology of sequence 6 and 56 and with the field MF MR which is thebzip field of a transcription factors containing a signal of nuclearlocalization and a trans-regulating activity in TAF1.

For the purposes of the invention, a preferred protein comprises atleast one sequence chosen from SEQ ID Nos 1-180 or a sequence having70%, 80% or preferably 90% homology with said sequence (SEQ ID No 215).

The LIV21 complex comprises proteins including 3D helices structures,which have a major functional role for their interactions with the restof the Liv21 complex.

The present invention concerns a purified or recombinant, isolated humanpolypeptide having a sequence comprising the sequence SEQ ID No 1 and/orSEQ 5 ID No 2 and/or SEQ ID No 3 and/or SEQ ID No 4 and/or SEQ ID No 5.Preferably, the polypeptide LIV21F comprises the sequences SEQ ID Nos 1,2 and 5. In a preferred embodiment, the complex is studied based on asequence selected among the peptide sequences obtained by MALDI (FIGS.3, 4 and 5). The invention also concerns the three peptides LIV21a (SEQID No 1), LIV21b (SEQ ID No 2) and LIV21e (SEQ ID No 5). It alsoconcerns peptides comprising at least 10 consecutive amino acids ofhuman LIV21, preferably at least 20, 30 or 50 consecutive amino acids ofLIV21 peptides (i.e. sequences list 1-215 in annexes).

The present invention also relates a polynucleotide encoding for thehuman protein Liv21, Liv21a and/or Liv21b, generally a polynucleotideencoding for a polypeptide according to the present invention. Thepolynucleotide encoding for Liv21F and this one encoding for LIV21K maybe an mRNA, a cDNA or a genomic DNA. The polynucleotides according tothe present invention may be isolated from cells and more particularlyfrom human cells or from human cDNA libraries. They can also be obtainedby a polymerase chain reaction (PCR) carried out on the total DNA of thecells or else by RT-PCR carried out on the total cellular RNAs or bychemical synthesis. Probes and primers described in the presentinvention may be used to isolate and/or prepare a polynucleotypeencoding for a protein of the Liv21F. It relates also a cloning orexpression vector comprising such polynucleotide.

Such vector may include the elements required for the expression(expression vector) and eventually for the secretion of the protein in ahost cell (signal peptide of secretion). Preferably the said vectorscomprise: a promoter, signals of initiation and termination oftranslation, as well as adapted regions for transcription regulation.The vector can be a plasmid, a cosmid, a BAC, a phage, a virus, orother. The invention relates to a host cell or a non-human transgenicanimal including a vector or a polynucleotide according to the presentinvention.

The invention also concerns derivatives of interest of LIV21F or LIV21Kwhich are for example proteins of merger in which is amalgamated withproteins markers like the GFP. In addition, the LIV21F or LIV21K proteinand all described peptides (i.e. patent in listing) can be marked by anyknown means of those skilled in the art. The siRNA too (SEQ ID No 91 to118).

The present invention also relates to an antibody which is linkedspecifically to a polypeptide according to the present invention,preferably human LIV21, a fragment or a derivative of this one. In aspecific mode of realization, the antibody is linked specifically to aLIV21a or LIV21b peptide or LIV21F or LIV21K polypeptides.

The method can in particular comprise the detection of the product ofexpression of two of these genes or of the three genes. Moreover, atleast one of the ratios LIV21/PKCS, LIV21/E2F4 and LIV21/E2F1 can bedetermined in the present method. This ratio can be determined in thecytoplasm and/or in the nucleus. Preferably, these ratios are determinedin the nucleus. Preferably, these ratios are compared with thoseobtained in a normal cell.

In one embodiment, the expression product of the genes is detected atthe mRNA level, it being possible for the mRNA to be detected by anymeans known to those skilled in the art.

Thus, the method according to the present invention also relates to thedetection of a polynucleotide encoding the human LIV21 protein or afragment thereof, for example LIV21a and/or LIV21b. The polynucleotideencoding LIV21 may be an mRNA, a cDNA or a genomic DNA. Thepolynucleotides may be isolated from cells of the biological sample.They may also be obtained by a polymerase chain reaction (PCR) carriedout on the total DNA of the cells or else by RT PCR carried out on thetotal RNA of the cells or polyA RNAs.

The mRNA may be detected by an RT PCR analysis. For this, the methoduses a pair of primers specific for the expression product to bedetected, in particular LIV21, PKCε, E2F1 or E2F4. The term “specificpair of primers” is intended to mean that at least one of the primers isspecific for the expression product to be detected, i.e. that this pairof primers makes it possible to specifically amplify a fragment of thedesired mRNA. Preferably, the RT PCR analysis carried out on nuclearand/or cytoplasmic extracts of the cells contained in the sample fromthe patient. Optionally, the RT PCR analysis may be a quantitativeanalysis. A pair of primers specific for LIV21 can be prepared on thebasis of the teachings of the present application. For example, the pairof primers may comprise the primers described in the sequences listing 5SEQ ID Nos 3 and 4, and additional sequences of the list obvious thusnumbered from 119 to 149 and 171 to 180 with optionally all theadditional nucleotidic sequences of the additional list.

The pairs of primers specific for PKCs, E2F1 and E2F4 are well known bythose skilled in the art (Caroli J S 2000; Mundle S D 2003; Stevaux 02002; Cheng T 2002; Opalka B 2002). The mRNA may also be detected byNorthern blotting analysis. For this, the method uses a probe specificfor the expression product to be detected, in particular LIV21, PKCs,E2F1 or E2F4. A probe specific for LIV21 can be prepared on the basis ofthe teachings of the present application. An example of a specific probecomprises the sequence SEQ ID No 5. Preferably, the Northern blottinganalysis is carried out on nuclear and/or cytoplasmic extracts of thecells contained in the sample from the patient. The nucleic probe islabelled. The oligonucleotide labelling technique is well known to thoseskilled in the art. The labelling of the probes according to theinvention can be carried out with radioactive elements or with nonradioactive molecules. Among the radioactive isotopes used, mention maybe made of ³²P, ³³P or ³H. The non radioactive entities are selectedfrom ligands such as biotin, avidin, streptavidin or digoxigenin,haptens, dyes and luminescent agents such as radioluminescent,chemoluminescent, bioluminescent, fluorescent or phosphorescent agents.The probes specific for PKCs, E2F1 and E2F4 are well known to thoseskilled in the art. In a preferred embodiment, the expression product ofthe genes is detected at the protein level. Preferably, the protein isdetected using a specific antibody. Thus, the method comprises a step ofbringing the cells of the biological sample into contact with ananti-human LIV21 antibody. The antibodies can be monoclonal orpoly-clonal. The antibody can for example be a serum anti-LIV21. Whenthe product of expression of one of the genes PKCs, E2F1 and E2F4 mustbe detected, the method can use antibodies specific for the PKCs, E2F1and E2F4 proteins, respectively. Polyclonal and monoclonal antibodiesdirected against PKCs, E2F1 and E2F4 are commercially available. By wayof example, mention may be made of, for PKCs, a rabbit polyclonalantibody (Santa Cruz Technology, sc-214), for E2F1, a rabbit polyclonalantibody (Santa Cruz Technology, sc-860), and for E2F4, a rabbitpolyclonal antibody (Santa Cruz Technology, sc-866). Preferably, theantibodies are labelled, directly or by means of a secondary antibody.The antibody labelling techniques are well known to those skilled in theart. In a specific embodiment, the protein can be detected by Westernblotting analysis. The Western blotting analysis can be carried out onnuclear and/or cytoplasmic extracts of the cells contained in the samplefrom the patient. Briefly, the proteins are migrated in a gel and thenblotted onto a membrane. This membrane is then incubated in the presenceof the antibodies and the binding of the antibodies is optionallyrevealed using labelled secondary antibodies. In another embodiment, theprotein is detected by immunohistochemistry, immunocytochemistry orimmuno-radiography. These techniques are well known to those skilled inthe art. The immunocytochemical analysis can be carried out on wholecells originating from the sample or which are derived there from, forexample by cell culture. It can also be carried out on isolated nuclei.The immunohistochemical analysis can be carried out on mammary, nervetissue sections etc. . . . By way of illustration, an immunocytochemicalanalysis can include the following steps. However, it is understood thatother preparatory methods can be carried out. Cells originating from thebiological 5 sample are cultured, preferably on slides (Lab Tek, Nunc,Germany), and then washed with buffer and fixed with paraformaldehyde(for example, 4%). A saturation step is preferably carried out byincubating the cells with buffer S (PBS-0.1% Triton X100-10% FCS). Thecells are then incubated with a primary antibody and are then washed andincubated with a fluorescent secondary antibody, if necessary. Thenuclei can be labelled with propidium iodide (Sigma). The slides aremounted in moviol for observation by fluorescence microscopy. Moreover,isolated nuclei sampled during a nuclear extraction can be fixed withparaformaldehyde (for example, 4%). The suspensions of nuclei aredeposited between a slide and cover slip and the observation is carriedout by fluorescence microscopy and by confocal microscopy. The primaryantibodies are, for example, rabbit antibodies and the secondaryantibodies are labelled antibodies directed against rabbit IgGs. Thebiological samples originate from a patient potentially suffering fromcancer or for whom it has been established that said patient issuffering from cancer. “Biological sample” is intended in particular tomean a sample of the biological fluid, living tissue, tissue fragment,mucosity, organ or organ fragment type, or any culture supernatantobtained by means of taking a sample. The method according to thepresent invention can comprise a step of taking a biological sample fromthe patient. The detection step can be carried out directly on a tissuesection of the sample, or on a culture of cells originating from thesample, or on total cell extracts, nuclear extracts and/or cytoplasmicextracts. In a specific embodiment of the method comprising thedetection of the product of expression of the PKCe gene, a significantincrease in PKCs is indicative of the presence of cancer cells. Morespecifically, the amount of PKCs in normal cells is compared with theamount of PKCs in the cells of the sample, and the significant increaseis determined by means of this comparison. The method according to thepresent invention can optionally comprise the measurement of theLIV21/PKCS content. This LIV21/PKCS ratio increases in the cytoplasmicfraction of cancer cells compared with normal cells. In another specificembodiment of the method comprising the detection of the product ofexpression of the E2F4 gene, the method comprises the detection of theassociation of LIV21 with the E2F4 protein, and a decrease in thisassociation is indicative of the presence of cancer cells. The detectionof the association of LIV21 with the E2F4 protein can be carried out byconcurrent detection of LIV21 and of F9F4. The method according to thepresent invention can optionally comprise the measurement of theE2F4/LIV21 content. This E2F4/LIV21 ratio decreases in the nucleus ofcancer cells compared with normal cells. In an additional embodiment ofthe method comprising the detection of the product of expression of theE2F1 gene, the presence of the E2F1 protein in the nucleus is indicativeof the presence of cancer cells. The method according to the presentinvention can optionally comprise the measurement of the E2F1/LIV21Fcontent. This E2F1/LIV21F ratio increases in the nuclear fraction ofcancer cells compared with normal cells. The method according to thepresent invention allows in particular the detection of metastasizedcancer, therapeutic monitoring and/or recurrences following treatmentand makes it possible to determine the degree of invasiveness of acancer or neurodegenerative disease or Alzheimer disease. Thespecificity of the detection can be related to the crossing over ofinformation obtained through the existence and the topography of LIV21by all imaging and spectroscopy means and obtained by combination withother known cancerological indicators via protein arrays or microarrays.Thus, the detection based on LIV21 can be combined with the detection ofother cancer markers, in particular breast cancer markers, known tothose skilled in the art and nerve cancers. In fact, the presentinvention concerns a method for the therapeutic monitoring of ananticancer treatment in a patient suffering from cancer, comprising theadministration of the anticancer treatment to said patient and thedetection of cancer cells in a biological sample from the patient,according to the method of the present invention. A decrease in cancercells will be indicative of the effectiveness of the treatment. Thedetection of cancer cells in a biological sample from the patient,according to the method of the present invention, can be carried outonce or several times over the course of the anticancer treatment orafter the anticancer treatment. Preferably, the biological sampleoriginates from the tissue affected by the cancer treated.

Moreover, the present invention also concerns a method for the detectionof recurrences subsequent to an anticancer treatment of a cancer in apatient, comprising the detection of cancer cells in a biological samplefrom the patient, according to the method of the present invention. Thedetection of cancer cells in a biological sample from the patient,according to the method of the present invention, can be carried outonce or several times after the anticancer treatment. The detection ofcancer cells is indicative of recurrences. Preferably, the biologicalsample originates from the tissue affected by the cancer treated. Thepresent invention also describes a kit for carrying out a methodaccording to the invention. More particularly, the invention concerns akit for the detection of cancer cells in a biological sample from apatient, comprising one or more elements selected from the groupconsisting of an antibody which binds specifically to human LIV21according to the present invention and an anti-LIV21 serum according tothe present invention, an oligonucleotide probe specific for the LIV21mRNA and a pair of primers specific for the LIV21 mRNA. In a preferredembodiment, the kit comprises antibodies, which bind specifically tohuman LIV21. In another preferred embodiment, the kit comprises anoligonucleotide probe specific for the LIV21 mRNA. It may also comprisea probe specific for a “housekeeping” gene. The kit according to thepresent invention can comprise reagents for the detection of anLIV21-antibody complex produced during an immunoreaction. Optionally,the kit according to the present invention also comprises means fordetecting the product of expression of at least one gene selected fromthe group consisting of the protein kinase C epsilon (PKCs) gene, theE2F1 gene and the E2F4 gene. This detection means can be antibodiesspecific for the protein, oligonucleotide probes specific for the mRNAconcerned and/or a pair of primers specific for the mRNA. The presentinvention also relates to a diagnostic composition comprising one ormore elements selected from the group consisting of an antibodyaccording to the present invention and a serum according to the presentinvention, an oligonucleotide probe specific for the LIV21 mRNA and apair of primers specific for the LIV21 mRNA.

Anticancer Therapy

In the context of an anticancer therapy, it is possible to envisionincreasing the amount of LIV21 present in the nucleus. For this, thenuclear localization in cancer cells of LIV21 expression could bepromoted. Liv21 being able to translocate in the nucleus by itself, onecould envisage the construction of an expression vector including apolynucleotide coding for human Liv21 in order to over express theseprotein in the cell nucleus for that we wish regulate the proliferation(SEQ ID No 215). The expression vector encoding Human Liv21 can beadministrate in vivo to the patient by any mean known by those skilledin the art, SEQ ID No 215 for example and inhibitor peptide of Liv 21.For example, the expression vector can be administrated as naked DNA(for example EP 465529). The microinjection, electroporation, phosphateof calcium precipitation techniques and formulations using nanocapsulesor liposomes are other techniques available (SEQ ID No 217). Theexpression vector may also be in the form of a recombinant virus,including, a polynucleotide encoding human Liv21 inserted into itsgenome. The viral vector can be selected for example from an adenovirus,a retrovirus, in particular a lentivirus, and a virus adeno-associated(AAV), a herpes virus (HSV), a cytomegalovirus (CMV), a vaccine virus,etc. . . . .

So advantageous, the recombinant virus is a defective virus.

Preferably, the expression vector permits a cellular targeting. So thisvector could target cancer cells or a particular cell type that isaffected by cancer. Targeting of particular cellular type can berealized by placing the polynucleotide coding Liv21 under the control ofa promoter tissue-specific. In another alternative the expression vectormay be targeted, for example, in association with a specific molecule ofa particular tissue or cancer cells, for example a specific antibody toa molecule expressed specifically by the particular tissue or the cellsof cancer. Also the choice of expression vector may also influence thecellular targeting. Indeed, if the vector is a virus, the virus tropismnatural or amended may also allow a certain target.

The present invention concerns a pharmaceutical composition comprising apolynucleotide encoding for Liv21 (SEQ ID No 215), more particularly anexpression vector coding for Liv21. It also concerns the use of apharmaceutical composition comprising a polynucleotide encoding forLiv21, in particular an expression vector encoding for Liv21 asmedicament. Preferably, the present invention concerns the use of apharmaceutical composition comprising a polynucleotide encoding forLiv21, in particular an expression vector encoding for Liv21, for thepreparation of a medicament for use in treating cancer. Finally, itconcerns a method for treating cancer in a patient, comprising theadministration to the cancer cells of a polynucleotide encoding forLiv21, Liv21 expression making it possible to reduce or abolish thecancerous phenotype of the treated cells. Preferably, cancer is selectedfrom breast cancer, bladder cancer, ovarian cancer, uterus cancer lungcancer, skin cancer, prostate cancer, colon cancer, a glioblastoma,without being limited thereto. Moreover, the nuclear localization ofLIV21 could be promoted, for example by decreasing the activity of PKCsin the cancer cells and by using HDAC inhibitors.

In another specific embodiment of anticancer therapy, it is possible toenvision decreasing the activity of PKCs in the cancer cells. Thisdecrease in activity can be produced by decreasing the activity of thePKCs protein or by decreasing its expression. A decrease in the activityof the PKCs protein can be obtained by administering PKCs-proteininhibitors to the cancer cells. The PKCs-protein inhibitors are wellknown to those skilled in the art. A decrease in the expression of thePKCs protein can be obtained by using antisenses or siRNA specific forthe PKCs gene. Kits are commercially available. Moreover, the techniquesconcerning inhibition by means of antisense or siRNA are well known tothose skilled in the art (Arya R 2004, Lee W 2004, Sen A 2004, Platet N1998, Hughes 1987) (SEQ ID No 215 and SEQ ID No 216).

The next siRNA can be also used:

GCUGAGGCAGGCAGAUCAUUUCAA ) UUCGACUCCGUCCGUCUAGUAAGAG-GUACCAUUUCACAACAACUUUCAA ) UUCAUGGUAAAGUGUUGUUGAAGAG-

The present invention therefore concerns a pharmaceutical compositioncomprising a PKCs-protein inhibitor. It also concerns the use of apharmaceutical composition comprising a PKCs-protein inhibitor as amedicament, in particular for the preparation of a medicament for use intreating cancer. Finally, it concerns a method for treating cancer in apatient, comprising the administration to the cancer cells of aPKCs-protein inhibitor, the pKCs-protein inhibitor making it possible toreduce or abolish the cancerous phenotype of the treated cells. In afirst embodiment, the PKCs-protein inhibitor decreases the activity ofthe PKCs protein. In a second embodiment, the PKCs-protein inhibitordecreases the expression of the PKCs protein. Preferably, cancer isselected from breast cancer, bladder cancer, ovarian cancer, uteruscancer lung cancer, skin cancer, prostate cancer, colon cancer, livercancer, a sarcoma, a leukaemia and glioblastoma, without being limitedthereto. PKC epsilon inhibitors are published and commercially availablefor other applications.

In the context of a therapy for a neurodegenerative disease, it ispossible to envision decreasing the amount of LIV21 present in thenucleus. For that, the Liv21 expression could be decreases or blocked innuclear of the diseased cells of neurodegeneration. The cells affectedby the neurodegenerative disease are generally neurons, motorneurons,etc.

In a preferred embodiment, the neurodegenerative disease is chosen fromAlzheimer's disease, Huntington's disease, Parkinson's disease andamyotrophic lateral sclerosis (ALS). The inhibition or the blocking ofLIV21 expression can be carried out by any means known to those skilledin the art. In particular, by way of illustration, mention may be madeof the antisense strategy, siRNA and ribozymes. Thus, an antisenseoligonucleotide or an expression vector encoding this antisenseoligonucleotide could be prepared and used to block the translation ofthe mRNA encoding LIV21F in vivo. Moreover, a ribozyme can be preparedfor cleaving and destroying, in vivo, the mRNA encoding LIV21. It isalso possible to envisage a triple-helix strategy in which anoligonucleotide is designed so as to hybridize with the gene encodingLIV21 and to thus block the transcription of this gene.

Moreover, for this, the nuclear localization of LIV21 could also behindered, for example by increasing the activity of PKCs in the cellsaffected by the neurodegenerative disease. In one particular therapeuticmethod against neurodegenerative diseases, it is possible to envisionincreasing PKC epsilon activity in the cells affected by theneurodegenerative disease. This increase in activity can be produced byincreasing the activity of the PKCs protein or by increasing itsexpression. An increase in the activity of the PKCs protein can beobtained by administering PKCs-protein activators to the cells affectedby the neurodegenerative disease. The PKCs-protein activators are wellknown to those skilled in the art (Toma 0 (2004), Activation of PKCs byDAG, AGPI: oleic acid, linoleic acid, arachidonic acid, etc. . . .Activation and proteolysis of PKCs in gonadotropic cells: Communication2004 by Macciano H, Junoy B, Mas J L, Drouva S V, UMR6544 Marseille). Anincrease in the expression of the PKCs protein can be obtained by usingexpression vectors encoding the PKCs protein and which make it possibleto overexpress it in the cells affected by the neurodegenerativedisease.

Thus, the present invention concerns a pharmaceutical compositioncomprising a PKCs-protein activator or an expression vector encoding thePKCs protein. It also concerns the use of a PKCs-protein activator or ofan expression vector encoding the PKCs protein, for the preparation of amedicament for use in the treatment of a neurodegenerative disease.

Screening Method

The invention concerns methods for the selection, identification,characterization or optimization of active compounds, which decreasecell proliferation, based on the measurement of the nuclear versuscytoplasmic localization of LIV21, or of the binding of the LIV21protein to the E2F4 protein.

In a first embodiment, the selection, the identification, thecharacterization or the optimization of active compounds of therapeuticinterest comprises bringing a candidate compound into contact with acell and determining the nuclear versus cytoplasmic localization of theLIV21 expression product. An increase in the nuclear localization ofLIV21 indicates that the candidate compound is active in terms ofdecreasing or abolishing cell proliferation. A decrease in the nuclearlocalization of LIV21 indicates that the candidate compound is active interms of treating or preventing a neurodegenerative disease.

In a second embodiment, the selection, the identification, thecharacterization or the optimization of active compounds of therapeuticinterest comprises bringing a candidate compound into contact with acell and determining the level of expression of the gene encoding thePKCs protein. A decrease in the expression of PKCs indicates that thecandidate compound is active in terms of decreasing or abolishing cellproliferation. An increase in the expression of PKCs indicates that thecandidate compound is active in terms of treating or preventing aneurodegenerative disease.

In a third embodiment, the selection, the identification, thecharacterization or the optimization of active compounds of therapeuticinterest comprises bringing a candidate compound into contact with acell and determining the level of LIV21/E2F4 complex. An increase in thelevel of LIV21/E2F4 complex indicates that the candidate compound isactive in terms of decreasing or abolishing cell proliferation. Adecrease in the level of LIV21/E2F4 complex indicates that the candidatecompound is active in terms of treating or preventing aneurodegenerative disease. In a fourth embodiment, the selection, theidentification, the characterization or the optimization of activecompounds of therapeutic interest comprises bringing a candidatecompound into contact with a cell and determining the level ofexpression of the gene encoding the E2F1 protein. A decrease in theexpression of E2F1 indicates that the candidate compound is active interms of decreasing or abolishing cell proliferation. An increase in theexpression of E2F1 indicates that the candidate compound is active interms of treating or preventing a neurodegenerative disease.

The invention also relates to a method of screening for a compoundcapable of interacting in vitro, directly or indirectly, with LIV21,characterized in that: in a first step, the candidate compound and LIV21are brought into contact and, in a second step, the complex formedbetween said candidate compound and LIV21 is detected by any appropriatemeans.

The present invention also relates to a method of screening for acompound capable of modulating (activating or inhibiting) the activityof the LIV21 protein, characterized in that: in a first step, cells of abiological sample expressing the LIV21 protein are brought into contactwith a candidate compound, in a second step, the effect of saidcandidate compound on the activity of said LIV21 protein is measured byany appropriate means, and in a third step, candidate compounds capableof modulating said activity are selected. The activity of LIV21 can, forexample, be estimated by means of evaluating the ability of the cell todivide, by measuring the expression of the E2F1 gene or by thecytoplasmic and/or nuclear localization of LIV21.

The candidate compound can be a protein, a peptide, a nucleic acid (DNAor RNA), a lipid, or an organic or inorganic compound. In particular,the candidate compound could be an antibody, an antisense, a ribozyme oran siRNA.

Other advantages and characteristics of the invention will appear in theexamples and the figures which follow, and which are given in a nonlimiting manner.

EXAMPLES Example 1 Extraction of Proteins of the Liv21 Complex. TheMCF-7 Cell Line

The MCF-7 line is a non clonal human line of breast denocarcinoma cells.During their differentiation induced by exogenous factors, these cellsdevelop a hypertrophy, membrane protrusions and a tendency to dissociatefrom one another. They acquire a secretory phenotype which ischaracterized by the appearance of numerous granules and of secretorycanaliculi.

In vivo, these cells are relatively non metastatic and this lowinvasiveness is thought to be due to a low constitutive activity of theprotein kinases C (PKCs) and to a relatively low level of expression ofprotein kinase C alpha.

This line is used in many studies on proliferation, differentiation andapoptosis. These studies use appropriate drugs, such as TNF for theinduction of apoptosis, or TPA (12-O-tetradecanoyl phorbol-13-10sumoate) for the induction of differentiation and therefore for thestudy of departure from the cell cycle.

Extraction of Proteins of the Liv21 Complex.

After culture cells MCF7 (ATCC passage 15) and cell extraction, 5 mg ofprotein are centrifuged after homogenization in 10 ml RIPA buffer,antiprotease added. These protein extracts went loop for seven hours ata peristaltic pump on a column of affinity (HITRAP NHS ACTIV HP 1×5 ml:Article and catalog 17071701) which was set Liv21 antibody. Wash 3times, 10 ml in RIPA buffer and then a half-eluting fractions of 500 nlin a buffer glycine PH2/HCL, then control gel SDS Page 10% (deposit of25 nl of the fraction) followed by a 25 western blot to verify.Revelation of a band on gel at 51 kD flanked by two other bands at 50and 52 kD respectively and a lower trace of a band 80 kD and 100 kD ingel mono dimensional (FIG. 1A). Moreover revelation of 12 spots between50 kD and 70 kD in bidimensional gel (figure IB and 2), idem cultures ofSHSY5Y (i.e. FIG. 7).

SEQ ID No 1 Peptide LIV21a RVYGPLTNPKPQ SEQ ID No 2Peptide LIV21a PLMIIHHLDDCPHSQALK SEQ ID No 3Peptide LIV21c SYMSMFLLLMAISCVLAK SEQ ID No 4 peptide LIV21d CYRSILHTKVSEQ ID No 5 Peptide Liv21b KFFVFALILALMLSMCGADSHAKR

Example 2 Mass Spectrometry

A mass spectrometry (MALDI) was realized for the LIV21 protein and itscomplex. The LIV21 protein (polynucleotide Liv21F) was digested withtrypsin. The peptides derived from the digestion are solubilized in asolvent: acetonitrile/water (1/1) containing 0.1% of TFA(trifluoroacetic acid). A saturated solution of thealpha-cyano-4-hydroxycinnamic matrix was prepared in the same solvent.The same volume of the two solutions was taken and mixed together and 1μl was deposited onto the MALDI plate for analysis. The massspectrometry showed that the LIV21 protein and its complex digested withtrypsin reveals hundred peptides following the band of gel extractedbetween 49 and 54 kD (i.e. FIGS. 3-5). The LIV21 protein wascharacterized by a molecular weight of 50 kD revealed by Westernblotting and by a two-dimensional SDS PAGE gel (FIG. 2). But we find aproduct of 100 kD at 130 kD which could be a Liv21 dimer. (I.edescription FIGS. 4, 5 and 6)

When it changes cell compartment and when it is sumoylated, the LIV21protein has a molecular weight of approximately 60 kD. When it isphosphorylated in the cytoplasm, it exhibits two forms which differ by afew kilobases. A doublet is then observed.

Example 3 Analysis of Sequences in the Proteomic Databases

Several peptides in the patent in listing of the patent characterize it:

SEQ ID No 51 Peptide LIV21b FVFALILALMLSMCG SEQ ID No 5Peptide LIV21b KFFVFALILALMLSMCGADSHAKR

For example, the inventor obtained a very significant score of 81,hoped: 0.0012 for the histatin 3-2 variant with 52% of overlapping ofsequences between the tested sample and histatin, this sequence SEQ IDNo5 is commune to Liv21 and HIS3-HUMAN (FIGS. 6 and 7). Using differentdatabase and a ppm which differed to 20 to 50 ppm, we obtain the samesequence for the commune part: SEQ ID No 51.

Example 4 Reverse Transcriptions:

After MCF7 cells (ATCC passage 15) had been thawed and cultured up to 200 million, they were trypsinized and frozen at −80° C. The RNA wasextracted from two pools of 50 million cells with the Nucleospin RNA Lkit (Macherey Nagel) ref. 740.962.20, resulting in a pool 1 of 318 μgand a second of 182 μg.

The poly A+ RNA was extracted from 313 μg of total RNA of pool 1 usingthe oligo Tex mRNA Midi kit (Qiagen) ref.70042.

I Reverse Transcription:

The RNA was reverse transcribed with the Fermentas Revert Aid H minus MMuLV Reverse Transcriptase, ref. EPO451 batch 1124, with 3.64 μg oftotal RNA and 0.45 μg of mRNA, according to the supplier's conditions,with an oligo dT primer. Reactions were carried out at 2 differenttemperatures at 45° C. and 55° C. so as to eliminate the RNA structuresthat may hinder reverse transcription.

II PCR

The PCRs were carried out with the reverse transcriptions as templates,initially with the primers Al+oligo dT. Nested PCRs were subsequentlycarried out on these first PCRs, with the primers Al+Splicing, Al+GDBR1,or ATG+Splicing, ATG+GDBR1.

PCR amplification was then carried out with the primers specific for thegenes to be detected, using the cDNAs obtained after oligo dT RT.

Enzyme: Fermentas Taq DNA polymerase. Thermocycler: Bio Rad iCycler.

The quality of the cDNAs was tested by amplification of GAPDH, b actinand Histone H3.3 housekeeping genes.

TABLE 1 Primers Amplified fragment Reference 5′-3′ sequence sizeHistone N 5′-gtg gta aag cac cca gga a-3′ 347 bp Histone I (reverse)5′-gct agc tgg atg tct ttt gc-3′ Hum GAPDH sense5′-TGA AGG TCG GAG TCA ACG G-3′ 983 bp Hum GAPDH antisense5′-CAT GTG GGC CAT GAG GTC-3′ Hum b-actin sense5′-GGA CTT CGA GCA AGA GATGG-3′ 234 bp Hum b-actin antisense5*-AGC ACT GTG TTG GCG TAC AG-3′ LIV21 (Al) 5*-TCCTATGCTTTGACTATTAG-3′LIV21 (A2) 5′-CCTGACATCCCTACATCACCGCA-3* odT ATG galgal5*-ATGTATATTATATCTAA-3′ Splicing sense histatin5′-TGTTGGGATTGCTTATATTT-3′ Splicing reverse histatin5′- AAATATAAGCAATCCC A AC A-3′ GDBR1 reverse 5′-CTTTATTATTTTGTAAAAT-3*Forward Reverse 25M Mg 10 mM primer primer 10× (1.5 mM final dNTP 10 uM10 uM Taq (ul) cDNA H₂O buffer concentration) (Ml) (Ml) (Ml) 5 U/ul (MO18.3 2.5 1.5 0.5 0.5 0.5 0.2 1 Other oligonucleotides (I e patent inlisting of Trail. PCR Cycles Denaturation: 94° C. 2 minutesDenaturation: 94° C. 3 0 seconds 5 Annealing: 52-55° C. 1 minute 35cycles Elongation: 72° C. 1 minute 3 0 Final elongation: 72° C. 7minutes Conservation: 4° C.

III Controls

The PCR Products were Subsequently Controlled on Agarose Gels AndAnalysed with the BIOCAPT 11.01 Software from Vilber Lourmat.

-   -   Gel 1 (ie FIG. 7): control of RNA on agarose gel 15 FIG. 8: RNA        pool

FIG. 9: PCR with housekeeping genes and analysis of molecular masses.

FIG. 10: PCR with the primers showing a band of 1400

-   -   bp. 20 FIG. 11: Gel 2 with analysis of molecular masses

FIG. 12: Gel 3 at 55° and analysis of molecular masses

FIG. 13: Gel 4 at 45° and at 55° and analysis of molecular masses. 25FIG. 14: screening ligation of 400 pb band, clones B1 to B10.

FIG. 15: screening ligation of 1400 pb band, clones CI to C10.

FIG. 16: Gel 5: ligation screening on the five new clones.

FIG. 17: Gel 6: Screening of the S55T and S55M recombinant clones andanalysis of molecular masses (i.e. ptenting listing for oligonucleotidicsequences).

Ge12: PCRs carried out using templates from PCRs performed with theprimers Al+oligo dT on the RTs carried out at 45° C. on the total RNAand the poly A+ RNA (messenger RNA). The primers used for these PCRs areAl+GDBR1 or Al+Splicing reverse.

On the RTs carried out using the total RNA, a band of 1178 1253 bp isamplified with, the primers Al+GDBR1 and Al+Splicing reverse. The poly ARNA was used to carry out the RT and is weakly observed at the size(FIG. 10) of 1400 by for amplification with the primers Al+G, and of 415bp with the primers Al+Splicing reverse. In the Al+splicing PCR product,there are other bands, of 860 and 233 bp (FIG. 11).

Gel 3: PCRs carried out using templates from PCRs performed with theprimers Al+oligo dT on the RTs carried out at 45° C. on the total RNAand the poly A+ RNA (messenger RNA). The primers used for these PCRs areAl+GDBR1 or 25 Al+Splicing reverse (FIG. 12).

For the PCRs carried out on RTs performed at 55° C., the same overallpattern of bands as that obtained on the RTs performed at 45° C. isfound.

No specific amplification is observed when the poly A RNA was used tocarry out the RT. On the RTs carried out on the total RNA, a major bandof 1554 1609 bp is found with the primers Al+GDBR1 and Al+Splicingreverse. A band at the theoretical size of 1455 bp is expected for anamplification with the primers Al+GDBR1 and a band with the theoreticalsize of 415 bp is expected with the primers Al+Splicing reverse. In the2 profiles, very clear bands of 1900-2100 bp and of 1000-1300 bp arefound, but with a weaker intensity than that of the band of 1500-1600bp.

In the Al+splicing reverse PCR product, there is another major band, of263 bp.

Ge14: Nested PCRs carried out using templates from PCRs 10 performedwith the primers Al+GDBR1 or Al+Splicing reverse on the RTs carried outat 45° C. on the total RNA and the poly A+ RNA (messenger RNA). Theprimers used for these PCRs are ATG+GDBR1 or ATG+Splicing reverse (FIG.13).

The nested PCRs carried out with the primers ATG+GDBR1 give bands at1213 bp (RT 45° C.) and 1559+1315 bp (RT at 55° C.); the expectedtheoretical size is 1455 bp. The PCRs carried with the primersATG+Splice reverse give more varied band profiles. These PCRs carriedout on other PCRs performed with the primers Al+GDBR1 or Al+Splicingreverse. The presence of a band of 400 bp is noted in the profilesobtained from messenger RNA (the band obtained from the reversetranscription carried out at 55° C. is of greater intensity).

The profiles of the ATG+Splice reverse PCRs carried with total RNA atthe start give a band of 424 437 bp of very strong intensity. Bands of614 and 783 bp of very strong intensity are also found in the profile ofthe RT 45 and a greater number of bands, but of weaker intensity, isfound in the profile of the RT 55, bands at 1118, 936 and 749 bp.

The products of these various PCRs were cloned and sequenced.

Example 5

The PCR products of lanes 2, 4, 6, 7 and 8 were ligated with the plasmidpGEMT Easy, Promega, and the recombinant clones were screened (FIG. 16).

Lane 2: G45T ligations Lane 4: S45T ligations Lane 6: G55T ligationsLane 7: S55M ligations Lane 8: S55T ligations

The recombinant clones obtained were screened (after extraction of theplasmid DNA) by restriction with the Eco RI enzyme, the sites of whichborder the site of insertion of the PCR products into the pGEMT Easyvector.

Screening of the Recombinant Clones:

The first experiments had been carried out using the ten clones B andthe ten clones C, FIGS. 14 and 15, and the results of the sequences ofclones B2 and C8 are given in the following example, and exhibit, bysequence comparison between clones, great homology with the clones ofthe second series of experiments.

Gel 5: screening of the S45T and G45T recombinant clones

Analysis of Molecular Masses

The screening of the clones with Eco RI shows that, out of the 9 S45Tclones, 3 have inserts of 100 bp, 216 bp and 410 bp. On the G45T clones,out of the 6 clones tested, 3 have inserts of 57, 71 and 148 bp.

FIG. 17: screening of the S55T and S55M recombinant clones

Analysis of Molecular Masses

The screening with Eco RI shows that, out of the 13 clones screened, 7have inserts of sizes between 239 and 637 bp. The clones G45T5 (148 bp),S45T9 (410 bp), S45T3 (100 bp), S55M1 (491 bp), S55T6 (251 bp) and S55T9(637 bp) were extracted so as to be sequenced. Primers used fordetermining the sequences of the 15 various clones and are described inthe patent listing:

ATG galgal 5′atgtatattatatatctaa 3′ INV COMP ttagatatataatatacatSplicing reverse 5′ AAATATAAGCAATCCCAACA 3′ Splicing reverse 5′TGTTGGGATTGCTTATATTT 3′ inv comp GDBR1 reverse 5′ CTTTATTATTTTGTAAAAT 3′GDBR1 reverse 5′ ATTTACAAAATAATAAAG 3′inverse complement Cloned fragments

The test of the bio-markers on extracts of neuroblastomas and cellcultures SHSY reveals by RT PCR the play of under expression and of onexpression following in this example of analysis: Example of 16biomarkers of interest analyzed by RT PCR which recuts the resultsobtained by biochip on sensor-chip making it possible to see by SPR theover expression and the under-expression of certain genes of the Liv21complex and its partners of interaction for example. An under expressionof DKK1, SKP2, DKK3, ID2, p21, SKP2, TP531NP1, ID2, P73 is observedwhereas an over expression of MYCN, ALK, NLRR1 (the neuronal leucinerich repeat is transactivated), CRABPII, DDX1, LIV21K, AURORA kinase Ais observed.

Example 6

From the cloning of the LIV21 gene described above, the new sequencesare studied in order to design the most specific and effective siRNAs(ie. listing of siRNAs and FIG. 19) for creating “silencing” of thegene, i.e. inhibiting its expression. In the knowledge that the effectof the inhibition at each injection of siRNA remains short, i.e. mostcommonly less than one hour, the inventor has developed diagnosticproducts and therapeutic products from this same tool, namely the siRNA.

Example 6.1

The inventor uses siRNAs labeled with rhodamine or fluorescein or anyother label that can be revealed and followed by optical observationwith a microscope in order to localize the site, in the cells, tissuesor sample labelled, that the labelled siRNA will go to in order toattach to the specific sequence which characterizes it, and thus toindicate the site of the expression of the messenger RNA of the gene ofinterest. Thus, the specific siRNA can be used as a diagnostic marker asan antibody would be, and can make it possible to localize, in aspecific case such as on extemporaneous samples or any other type ofsample taken from a patient, for example a cancerous tissue sample, thefluorescence or any other labelling used on the siRNA and found in acell compartment on the sample.

Thus, a labelled siRNA of LIV21 (FIG. 19) that is found, under amicroscope, in the cell nuclei at the periphery of a surgical exeresis,would indicate a diagnosis of complete exeresis of the cancerous tissue;on the other hand, this same marker found in the cytoplasm or themembranes of this same sample would mean that the surgeon would have toperform an enlarged exeresis, immediately if this test can be carriedout directly in the operating theatre, which would be the best situationfor removing all the cancer cells visible only on this molecular andcellular scale by virtue is the siRNA marker. This could be a more rapidimplementation alternative than the other application that the inventorhas developed with the antibody or the LIV21 peptide, which can be usedin the same manner.

Example 6.2

For the treatment products which rise directly from the observationsmade thanks to the results of expression of the biomarkers on thebiochips, the interest is well illustrated for the example of the TGFBéta and p27:

The publication of Mr. Lecanda J and Gold Li of March 2009 is very wellrich of teaching and famous all the interest of the pharmaco diagnosticbiochips as described above.

TGF Béta inhibits cell proliferation by increasing the level of p27 viathe activation of the factors of transcription smad 2/3.

TGF Béta thus increases the total level of translocation of p27 in thecore accumulates p27 in the cellular core through the activation of smad2 and smad 3.

All these events are locked by an inhibiter of TGF Béta IH: serinekinase SD208: new centers therapeutic targeted for the carriers.

TGF Beta decreases the level of the components skp2 compared to proteinbut not the level of mRNA.

TGF Béta thus can mediate the degradation of p27 in the proteasome atthe proteinic level because it maintains it in the core but on the otherhand the inhibition of the transcription of p27 with a specific siRNAlocks TGF beta.

TGFBeta prevents degradation proteasomale kinase cycles dependantinhibiter on p27. (Alvarez Rodriguez R Pon S J that sci 2009 Mar.1-122).

The gene expression proneural coding for Mash 1 abolishes MYCN (mitoticactivity). (2009 Liu W Wuz Guan M, Lu Y).

The inventor uses first labelled siRNAs of the Liv21 complex in order toevidence their expected presence in the cellular compartment and tovisualize them. Then the inventor will use non-labelled Liv21 siRNAssolely for their therapeutic role. In a specific case, the injection ofsiRNA (FIG. 19) directly in a neurodegenerating tissue or any other meanof administration allowing to the siRNA to get to the neurodegeneratingtissue (for example the ear, the eye, cephalorachidian liquid, etc.) andto act allowing proliferation until apoptosis and therefore the death ofthe neurodegenerating cell.

An experiment on human cell cultures then on animal models having aretinopathy generating a vitreo-retinal degeneration (direct injectionof the siRNA in the eye) makes it possible to illustrate this method.

The siRNA are also transfected in the cells according to the Invitrogenprotocol using the lipofectamine 2000.

The study of the specific role of the PKC epsilon on the nucleartranslocation of Liv 21F can use a peptide which inhibits function PKCepsilon and the translocation like previously described but alsoaccording to the protocol of reference of Si RNA of PKC epsilon. Theprofile of expression observed corresponds to a doublet of tapes in thecytoplasm with approximately 51 KD (and with a tape of 64 kD accordingto the siRNA used) whereas it corresponds only to one simple tape with50 kD in the nuclear fraction.

The molecules of siRNA resulting from the nucleotidic sequences of theLiv21 complex can be selected in the group made up of the molecules ofsiRNA understanding any of sequences SEQ ID No 119 to 126 and 171 to175, and SEQ ID No—the use of a multitherapy based on the combination ofseveral of these siRNA for the processing of cancerous orneurodegenerative pathology. These siRNA and the useful combinationdeduced from the results from on expression and from under expression ofthe biochips and RT PCR constitute an auxiliary processing of pathologyand can be combined with small doses of chemical molecules havingalready proven reliable in the processing of pathology.

Example 6.3

The present invention, relates to methods and reagents useful inmodulating gene expression associated with Alzheimer disease in avariety of applications, including use in validating therapeutic,diagnostic, target, and applications genomic discovery.

The invention concerns compounds, compositions and methods for thestudy, diagnosis and treatment of diseases that respond to themodulation of beta secretase (BACE) amyloid, expression and/or activityof protein gene the precursor APP, of pin 1 of presenillin 1 (PS-1)and/or of presenillin 2 (PS-2) in association with modulation of geneand protein complex Liv21. The invention concerns compounds,compositions and methods for the study, diagnosis and treatment ofdiseases that respond to the modulation of the expression and/oractivity of genes involved in complex liv21 and in combination with thegenes of the betasecretase (BACE), amyloid protein of precursor APP,pin-1, presenillin 1 (PS-1) and/or presenillin 2 (PS-2).

Specifically, the invention relates to small nucleic acid molecules,such as short nucleic acid interferent (siRNA), bicatener RNA (dsRNA),micro RNA (miRNA), short double spin RNA (shRNA) be able to interferewith RNA of mediation (RNAi) against beta secretase (BACE) mediation,gene expression of the protein precursor APP, of pin-1, of presenillin(PS-1) and/or presenillin 2 (PS-2).

Vectors to Produce siRNAs are Described.

Vectors are also commercially available. For example, the psilencer isavailable in Gene Therapy System, Inc. and the RNAi system of pSUPER isavailable in Oligoengine. They deliver compositions of siRNA with codingRNA part.

Xia et all 2002 Zeng et all 2002Thijn et all 2002 Lee et allBiotechnology 19 Nature Mc Manus et all.

Sui et all PNAS 2002, Yu et all 2002 PNAS 99. Shi et all 2003.

The pharmaceutical compositions comprise a pharmaceutically acceptablecarrier.

The compositions are also provided with a device for administering thecomposition in a cell or in a subject or a tumor. For example, acomposition may be in a syringe or to a stent.

Example 7 Pharmaco-Diagnostic Test

Based on the observations that follow in annex at the end of thedescription of the example concerning the properties of the Liv21complex, the inventor conceived a design for a pharmacodiagnostic testclinically applicable by known technologically means, which can differaccording to users and correspond for each mean to a new product. Theinvention consists in the fabrication of diagnostic DNA, protein andantibody arrays, including the antibodies already known for thedifferent proteins of the complex associated with Liv21 according to thephase of the cell cycle, that is the antibodies, peptides or nucleotidesequences of the following genes: RBP2, E2F4, E2F1, SUMO, INT2, CRB2,HDAC1, TGFbeta, integrin_alpha5 beta2, Myob, MyoD, cycE/cdk2, cdk1,chk1, chk2, TNFalpha, CREB 1 and p300, Rb, p107, p130 from the pocketprotein family. But also NFkB, cdc2A, mdm2, p21, p53, p65, p73 RAS,Ki67, CAF1. The protein arrays (FIG. 20) will allow the study of over orunder expression of the gene products, the protein interactions and thepost-translational modifications, more particularly phosphorylations andmethylations of certain proteins, which indicate a specific state of theunhealthy cell that is different form the protein interactions and themetabolism of an healthy cell. The expression and silencing state ofcertain genes is different.

Example 7.1

pharmacodiagnostic biochip (FIG. 20) conceived based on nucleotide orpeptide sequences fixed on classical supports and according to knowntechniques such as Agilent or Affymetrix or Caliper without restrictionthereof and corresponding to known sequences of following genes andproteins listed in the patent, preferably using sequences that, by 3Danalysis, show preferably a loop or helix-loop-helix or basic loop orzinc finger 3D structure or a 3D conformation similar to an helixcorresponding in most cases to functional sites, or nucleotide sequencescorresponding to a region of cystein methylation, methylation of thepromoter region of the gene inducing a potential silencing (see generalbibliography), as well as new sequences of Liv21F et Liv21K listed inthe description and in annex, but also sequences common to certain genesand certain virus thought to be implicated in particular in breastcancer of Chinese population:

Mdm2 and HIV 1:

GAVTSSNIAA; DLDQSV; EGF and HPV16; EWWRLD; KNSLD; MHIESLDS; BCAS3; BCAS4

These sequences may be tested by microfluidic techniques upon fixationon a gold coverslip in order to study the protein interactions with thecell extracts of the patient.

But which can also be studied by a MICAM technique implemented of thebiochips which (cavities) bio-markers by piezo electric effect and notby physicochemical fixation.

Example 7.2

microfluidic test, for example type Biacore, using the SPR (plasmonicsurface resonance) technique known by those skilled in the art based ona support fixed with a gold film, which allows, once the light beam hasbeen sent to the interface, to obtain an adsorbed energy as a functionof the presence and the size of protein complexes (in the case ofprotein protein or protein antibody interactions) or of protein DNAcomplexes (in the case of protein DNA interactions) or RNA, or proteinor peptide and an evanescent wave perpendicular to the interface axis(use of a prism). The inventor fixes the selected peptide or DNAsequences on gold particles and calculates the rU number as a functionof the size of molecules cited in this patent for each interactioncomplex studied. In microfluidic, the liquid flowing over these goldfilm arrays may be a cell extract (or a selective one, meaning that itis only made of cell nuclei, or solely cell cytoplasms or solelycytoskeletal proteins etc.) according to extraction, separation andfractionation techniques such as Calbiochem: subcellular proteomeextraction or tissue or cell extracts without any other preparation thananti-proteases or cephalorachidian liquid or serum issued from a patientsample in order to study the circling marker (i.e. described nucleotideor peptide sequence list and/or known sequences of genes involved in theproliferation cycle listed).

Example 8

biochip of sequences SEQ ID No 119 to 127 and 180 with compounds withthe sequences of microRNA of interest in studied cancer: mir 21, mir34a, etc. . . . without the biochip being limited to these. Thetechnique of fixing of RNUMS on the supports defers according to theform of mediums, of revelation and biochip used. For example one usesthe sequences RNUMS quoted in solution biothynilées then fixed onsensor-chips at the streptavidine, if one tests their interactions withthe cellular or plasmatic extracts or of the rachidian fluid cephalopassing in the channels (fluidic microcomputer) of a A100 or T100. Itcan also be used as described in the US patent 2008 045418 A1 RNAslocked in end 3′ and added a ligase T4 RNA and a “labeled nucleic acidadapter” having a residue 5′ phosphates.

Example 9 Study of the Expression of LIV21 in Breast Cancer and ColonCancer Biopsies

In order to determine whether the observations obtained above areapplicable to human tissues, a large number of cancer biopsies obtainedfrom patients were studied by immunohistochemical reaction with LIV21specific antibodies. The immunohistochemical determination of LIV21protein expression was carried out on several biopsies from patients.Moreover, some paraffin slides from patients suffering from bladdercancer and from breast cancer were also studied. The improvement of theinvention is due to the fact of standardizing the biopsies or thepunctures by a technique of taking away standardized and gauged in orderto then allow reliable comparisons between cellular extracts of patientsand witnesses or between cytoplasmic and nuclear and membrane fractions.This same standardization implemented to the taking away making itpossible to compare the results of overexpression or of underexpressions of the bio-markers by RTPCR is essential to obtain reliableand comparable results.

Immunocytochemical Analysis Protocol:

1) Deparaffinize the slides, Rehydrate the tissues.2) Saturate the nonspecific sites and permeabilize the membranes.3) Add the antibody in a humid chamber, Reveal the antibody.

Deparaffinize the slides under a hood.

Two successive baths of toluene (rectapur Prolabo) 2×30′ min or 2×20min; then dehydrate the tissues with rectapur alcohol at 100% for 15min; then rectapur ethanol at 95% for 10 min; then rectapur 70% for afurther 10 min.

Thaw the antibody at the same time. Rehydrate the tissues gently in PBSsupplemented with 10% fetal calf serum and 0.1% Triton.

Saturate the nonspecific sites (for example, with ovalbumin) andpermeabilize the membranes, Rehydrate for one hour.

Deposit one ml of this “PBS” per section in order to cover the slidewithout it drying out at any moment (when it is a slide with cells andnot tissues, half an hour is sufficient).

Place the pane and the stainless steel cover and water below so as tocreate a humid chamber.

Add the antibody in the humid chamber.

Dilute the rabbit serum to 1/200 in 4 ml of PBS triton, so as tocontinue to permeabilize the membranes and FCS.

Place 1 ml on each slide and keep away from the light and avoidevaporation. Leave overnight or for a minimum of three hours.

Then rinse with 1× normal PBS pH 7, carry out two washes of 5 to 10 minso that no trace of the first antibody remains.

While preparing the Alexa 488 green probe (in the cold at 4° and in thedark) diluted to 1/250, therefore 10 μliter in 2.5 ml of PBS, still with10% FCS and 0.1% Triton,

Rest the slides on the plate. Cover the section again with 2.5 ml inorder to maintain a humid chamber for one hour, and then wash with 1×PBSpH 7.

Wash with propidium iodide at 0.5 microgram per microliter to be dilutedto 20 microgram per ml and then again to 1/50, but this time, diluted in1×PBS alone (50 microliters per 2.5 ml of PBS). Drain while taking themout of the PBS and then dispense 2.5 ml of propidium iodide over thefour slides for one minute, followed by two rinses with simple PBS.Mount the slides in Moviol before reading.

For an immunolabelling with peroxidase it is mandatory to mask theantigenic sites by a 20 minutes water bath step in order to obtainmeaningful results when one is working with paraffined coverslips.

All these results show that the cytoplasmic localization of LIV21 is anindicator of the aggressiveness and of the metastatic potential of thecancer. The detection of LIV21 expression indicates the presence ofinvasive, aggressive and metastatic cancer cells. These results alsoshow that the nuclear localization of LIV21 is an indicator of normalquiescent cells, that is of well-differentiated tissues.

Annex: Study of the Nuclear Translocation of LIV21 in MCF-7 and SHSY5YCells

The study of the subcellular distribution of LIV21 in different tumorlines of various origins showed an plasmonic surface resonanceexclusively cytoplasmic localization of this protein.

The presence of putative phosphorylation sites by protein kinases C(PKCs) in the Liv21 sequence directed the study toward an involvement ofthese proteins with respect to its nuclear translocation. The MCF-7 linetreated with TPA modulates PKCs and is used in the present study.

The Effect of TPA on the MCF-7 Line

TPA is a known activator of PKCs. It activates the growth of normalbreast cells, does not modify the proliferation of the cells of benigntumors from this same tissue, but drastically inhibits the proliferationof the cells of human mammary tumor lines such as the MCF-7 line. Itreduces the cell growth of this line by positively controlling thec-erb-2 receptor and negatively controlling the retinoic acid receptora, which are both expressed in particularly large amount in these cells.The TPA greatly and rapidly inhibits the expression and the function ofestrogen receptors (ERs) and it induces the time- and dose-dependenttranslocation of protein kinases C (PKCs) from the cytosol to themembranes. Furthermore, TPA increases the migratory capacity of MCF-7cells in vitro and a short period of treatment of these cells with TPAinduces cellular expansion and microtubule organization characteristicof their differentiation.

Expression of LIV21 in MCF-7 Cells and in the SHSY5Y Cells

Firstly, the inventor verified the expression of LIV21 in these cells atthe transcriptional level and at the protein level.

The expression of the LIV21 mRNA was demonstrated by RT-PCR (senseprimer: (CCTGACATCCCTACATCACCCAT) (SEQ ID No 3, SEQ ID No 217 and SEQ IDNo 127 to 171) and antisense primer: (TCCTATGCTTGACTATTGC) (SEQ ID No 4)in these cells compared with the cells from breast tissues (FIG. 2A). AnmRNA of the same size as the mRNA detected in the breast tissues andspecific for LIV21 was detected. However, the level of expression ofLIV21 in the MCF 7 cells is lower than in the breast tissues. This firstresult shows that the MCF 7 line expresses the LIV21 mRNA.

The inventor tackled the study of the expression of the LIV21 proteinthrough the Western blotting technique, with an anti-LIV21 antibody, inMCF-7 cells compared with mammary tissues. The anti-LIV21 antibodieswere obtained by the method described below. In this line, LIV21 isexpressed, both in the mammary tissues and in the MCF-7 cells, in theform of a doublet which migrates at an apparent molecular weight of 51kDa.

Production of Purified Anti-LIV21 Serum

The specific peptide sequences are the sequences No. 1 and No. 2 and No.180.

These peptides were injected into rabbits (NZ W ESD 75 female, 2.3 kg atday 0), in agreement with standard immunization procedures,

The effect of the inhibitors on the cells of neuroblastomas

Study of the Influence of PKCs on the Nuclear Translocation of LIV21

Effect of TPA on PKC Expression

Western blotting study: Given that the protein sequence of LIV21 hasputative PKC phosphorylation sites, including two specific for PKC, theinventor tested the variation in the expression of this PKC as afunction of the duration of TPA treatment. It was observed that TPA actsvery rapidly on PKC expression, which decreases from 30 min. Theexpression of PKCzeta (PKCζ) is used as an internal control since it isnot sensitive to TPA.

P65L26 Immunocyto . . . P66L4 Nucleaire de LIV21

Study of the specific role of PKC on the nuclear translocation of LIV21using an inhibiting peptide of the function and translocation of PKC.

To determine the specific action of PKC on the translocation of LIV21,the crops were discussed with a peptide, selective antagonist of thefunction and translocation of this PKC (EAVSLKPT (SEQ ID No 6), and theresults were compared with those obtained by processing with TP A. Thispeptide is recognized by the enzyme and links themselves as a substrateamended to the level of its catalytic site. Not phosphorylable, he actsas a specific inhibiter of the activity of PKC.

The effect of the selective inhibition of the activity of PKC on thenuclear translocation of LIV21 was studied by immunocytochemistry. Theseexperiments were carried out on nontreated cultures or cultures treatedfor 12 h with TPA at 25 nM or with the peptide at two differentconcentrations, 1 and 2 μM (the peptide used at the concentration of 2μM has an effect identical to that of TPA on the nuclear translocationof LIV21).

These results were supported by cell fractionation experiments oncultures treated with the PKC inhibiting peptide at 2 μM, compared withTPA-treated cultures. The same LIV21 expression profile was observed inthe form of a doublet in the cytoplasm and of a single band in thenuclear fraction.

The specific inhibition of PKC induces a nuclear translocation of LIV21,which suggests that LIV21 could be the target of PKC, which wouldmaintain it in the cytoplasm in a phosphorylated form.

Example Western Blotting Analysis

This example describes the conditions used for a Western blottinganalysis of cancerous brain cells.

The protein extracts are heated at 80° C. for 5 minutes in a Laemmlibuffer (pH 7.4, 0.06 M Tris, 3% SDS, 10% 3 5 glycerol, 1 mM PMSF,β-mercaptoethanol). The migration is carried out by SDS-PAGE (sodiumdodecyl sulfate-polyacrylamide gel electrophoresis). 10 to 20 μg ofproteins migrate in a 12% polyacrylamide gel for 1 h under denaturingconditions (migration buffer: 25 mM Tris base, 192 mM glycine, 1% SDS,pH 8.3). The proteins are then transferred onto a nitrocellulosemembrane (Schleicher & Schuell) for one hour by liquid 5 transfer, in atransfer membrane (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3).The membranes, saturated in PBS-0.1% Tween-0.1% Triton X100-5% skimmedmilk for one hour, are brought into contact with the primary antibodydiluted in PBS-0.1% Tween-0.1% Triton X100-1% milk at ambienttemperature with gentle agitation for one hour to two hours. Afterwashing, the peroxidase-coupled secondary antibody is incubated with themembranes for 1 h. Revelation is carried out by means of achemiluminescence reaction using the ECL kit according to the supplier'sprotocol (Amersham).

The primary antibodies used are:

The anti-LIV21 serum which was produced using two synthetic peptidesbased on the sequence of LIV21F: peptide LIV21a (SEQ ID No 1) andpeptide LIV21b (SEQ ID No 2) and/or peptide Liv21e (SEQ ID No 51). Thepeptides were coupled to hemocyanin before being injected into rabbitsfor the immunization. The polyclonal antibody was obtained from thesetwo peptides by having immunized two rabbits and having bled one rabbitso as to have a preimmune serum (in order to be sure that this antibodydid not already exist in this rabbit).

The rabbit anti-CDK2 polyclonal antibody (Santa-Cruz technology sc-163)diluted to 1/200.

The mouse anti-p21 monoclonal antibody (Dako, M72 02) diluted to 1/150.

The mouse anti-p27 monoclonal antibody (Santa-Cruz technology sc-1641)diluted to 1/100.

It was shown before that protein LIV21 is associated with bodies PML andthat, at the time of the sumoylation, LIV21 passes from a molecularweight of 50 kd to 60 kd. By immuno-precipitation, the inventor showed aCo-localization of L1V21 with SUMO in complex PML—SUMO/LIV21. (FIG. 12)

Antitumor role of PML bodies: At the proliferation stage, there arevisualized modifications in the PML bodies since these PML bodiesdissociate and degrade: (speckles), proteins then become available inthe nucleus for ensuring transcription, proliferation, immune reactionsand everything that is required for gene transcription. It has beenshown that PML associates with SUMO and with HDAC-1 (histonedeacetylase 1) and that its complex acts on the expression of E2F1 andPML thus acts on the arrest of proliferation by blocking E2F1. Thus, thePML/HDAC-1 complex down-regulates E2F1 expression. PML associated withRb (p130) binds to the deacetylated histones and blocks E2F1 by bindingto the chromatin.

In acute promyelocytic leukemias, PML is truncated and becomes a fusionprotein with the retinoic acid receptor. This fusion protein(PMLRARalpha) is due to a 15/17 chromosomal translocation. A newtreatment for this disease by combining arsenic and retinoic acid inorder to induce cancer cells into apoptosis has been reported in theliterature. The PML protein is thought to regulate proliferation incancers and lymphomas. The inventor has shown, by immunoprecipitation,the association SUMO-PML in which LIV21 is located.

In the above patents, it was shown that LIV21 is phosphorylated by PKC.The TPA-treated MCF-7 lines show an inhibition of cancerousproliferation and a cell differentiation, and LIV21 is translocated intothe nucleus. If a PKC-specific inhibitory peptide was used, it was theactivity and not the expression of PKC which was inhibited.

During this TPA treatment (25 nM), when E2F4, p130 and LIV21 werestudied (green fluorescence) in the nuclei labelled (DNA) with propidiumiodide (red fluorescence), the following were observed:

After 12 h, intranuclear green fluorescence signals with the samepattern for E2F4, p130 and LIV21;

After 48 h, when the proliferation begins, E2F4 has a comparablelocalization; but at 72 h, it disappears from the nucleus (to thebenefit of E2F1).

By observing, by double labeling, the co-localization of PML and ofLIV21 at 24 h of PKC treatment (i.e. merge: yellow fluorescence), it wasobserved that they are co-localized in the nuclei. At 48 h, theco-localization between LIV21 and SUMO (i.e.). The hypothesis is thatSUMO, which binds to LIV21, in fact targets LIV21 into the PML bodiesand that LIV21 is involved in the PML/SUMO/Rb/HDAC-1 complexes. LIV21 isphysically associated with PML and SUMO in the nuclear bodies, byimmunoprecipitation and by colocalization by immuno cytochemistry (Rb,p130 and p107 are pocket proteins which have the same binding site). TheRb proteins repress cell growth (Fabbro, Regazzi R, Bioch Biophys ResComm 1986 Feb. 2; 135 (1): 65-73).

Physical Interaction of LIV21 with the Proteins of the E2F Family

Coimmunoprecipitation experiments carried out using anti-LIV21,anti-E2F1 and anti-E2F4 antibodies made it possible to demonstrate thatLIV21 associates with E2F4.

It was shown that BRCA1 also re-entered in interaction with familymembers E2F and thus complex LIV21. Indeed, E2F1 interacts with BRCA1and Brca1 interacts with MYC, ESR1, CHEK1 and MAP3K3.

The members of the E2F family are transcription factors whose role hasbeen widely described in the literature as being key molecules in thepositive or negative control of the cell cycle (Slansky JE and FarnhamPJ 1996; Helin K 1998 and Yamasaki L, 1998), by virtue of theirassociation with the pRb protein (WuCL, Zukerberg LR) or pocketproteins. E2F1 positively controls the cell cycle by transactivating 3 5the promoter of the genes responsible for cell proliferation (DNApolymerase alpha, thymidine kinase, DHFR, etc.), whereas E2F4 isdescribed as one of the members of the EF family which negativelycontrols the cycle. Furthermore, a high expression of E2F1 in embryonicmammary tissues has been shown (Espanel X, Gillet G 1998), whereas it isno longer expressed in post-mitotic mammary tissues, to the benefit of alarge increase in E2F4 expression (Kastner A Brun G 1998).

The identification of antigens has been carried out in cell lysates byimmunoprecipitation. The analysis of the physical interaction of variousproteins associated with E2F4 and E2F1 was demonstrated bycoimmuno-precipitation of protein complexes. The complex was studiedusing U. MACS PROTEIN with MICROBEADS (MILTENYIBIOTEC). When lysates ofS aureus are added, the proteins A interact with the Fc portion of thespecific antibodies and the immunocomplexes become insoluble and aretherefore recovered by centrifugation. After breaking of the bonds(heating) between AG/AC and protein A-rich membranes, Western blottingwas carried out. These results suggest that the LIV21/E2F4 complexappears to play an important Role in establishing cell quiescence. Astudy of co immunoprecipitation is following also with the profoundmammalian kit Pierce.

Functional interaction of LIV21 with the proteins of the E2F family

It was demonstrated that blocking the expression of the LIV21 proteinwas correlated with a decrease in the expression of E2F4 and with anincrease in the expression of E2F1. In parallel, the functional aspectof the increase in E2F1 was verified by studying the transcription oftwo of its target genes, DHFR and DNA polymerase a.

In conclusion, these results suggest that the LIV21/E2F4 complex acts asa complex which inhibits the expression of the E2F1 gene. This complexcould 3 5 correspond to a new point of control in the arrest of cellproliferation.

REFERENCES

-   Arya R, Kedar V, Hwang J R, McDonough H, Li H H, Taylor J,    Patterson C. Muscle ring finger protein-1 inhibits PKC{epsilon}    activation and prevents cardiomyocyte hypertrophy. J Cell Biol. 2004    Dec. 20; 167(6): 1147-59. Epub 2004 December-   Aurelian Radu, Nicolae Ghinea (2010)-   Expression of FSH receptor in tuymor blood vessels N England J Med    (363:1621-16802010)-   Stevaux O, Dyson N J. A revised picture of the E2F transcriptional    network and RB function. (2002) Curr Opin Cell Biol, 14 (6): 684-91.-   Yamasaki L. Growth regulation by the E2F and DP transcription factor    families. (1998) Results Probl Cell Differ, 22:199-227.

1. A Human Liv 21 complex characterized in that it comprises: anucleotide sequences selected from SEQ ID No 171 to 175 and SEQ ID 217an siRNA sequence derived from one of the RNA sequences seq120 and 121and 171 to 175 and all siRNA sequences (SEQ ID 91 to 118) a proteinfraction comprising at least sequence SEQ ID 1 to 181 or a sequencehaving 90%, and preferably 80%, and more preferably 70% identity withsaid SEQ ID 1 to 181 and SEQ ID No 183 and SEQ ID No 215 to 220 or asequence having 70, 80 or 90% identity with said sequence SEQ ID 183 and215 to 220 and SEQ ID
 230. 2. Liv 21 human complex according to claim 1,further comprising: A nucleotide sequences SEQ ID No 123, 124, 127 andthe protein fractions comprising at least sequence SEQ ID No. 1 or asequence having 70, 80 or 90% identity with said sequence SEQ ID No 1and the sequences 181 to
 185. 3. The Liv21 human complex is furthercomprising: The nucleotide sequences of any one or ribonucleotidesequence SEQ ID No 119 to 126 or 127, or a sequence having 90%, andpreferably 80%, and more preferably 70% identity with said sequence SEQID No 119 to 127, or a sequence having 90%, and preferably 80%, and morepreferably 70% identity with said sequence SEQ ID No 119 to 127, orUUGGUAACGACCAUGCCAC or UUCACUUAGAAUAAUGUCC or UCUUUGUGAAUUUGACAAC orUCAAGGUCCAGGCUACAAC or any of the following siRNA sequences SEQ ID No 60to 88 and 92 to 118, and GUGGCAUGGUCGUUACCAA dTdT DTdTCACCGUACCAGCAAUGGUU GGACAUUAUUCUAAGUGAA dTdT
 4. Liv21 human complexaccording to claim 1, further comprises at least one of: Any one of thenucleotide sequences SEQ ID No. 123, 124 and SEQ ID No 127 to 149 or Anyone of amino acid sequences SEQ ID No 1 to 118 and SEQ ID No 150 to 170and SEQ ID No 180 to 185: or a sequence having 90%, and preferably 80%,and more preferably 70% identity with said sequences SEQ ID No: 1 to185.
 5. Liv21 human complex according to any one of claims 1 to 3,characterized in that it interacts with at least one of the partnersassociated, said at least one of these being associated partnersselected from the group consisting of; any of the following proteins:RBP2, TNF alpha crb2, cmd2, cycE/cdk2, cdk1, CREB1 and p300, Rb, p107,p130 of family of pocket proteins, NFkB, cdc2A, mdm2, p21, p53, p65,p73, CyclinA and D1, BCAS3, BCAS4, Solute Carrier; or a antibody of anyone of the following proteins: RBP2, E2F4, E2F1, E2F2, SUMO, HDAC1,crb2, Int2, cmd2, cycE/cdk2, cdk1, CREB1 and p300, Rb, p107, p130 offamily of pocket proteins, NFkB, cdc2A, mdm2, p21, p53, p65, Ki67, CAF1,CyclinA and D1, CHUK, BCAS3, BCAS4, Solute Carrier.
 6. Method ofdetecting the complex human Liv21, characterized in that it comprisesthe implementation of at least one probe specific for at least onesequence of said human Liv21 complex according to claim
 1. 7. Method ofdetecting the complex human Liv21, characterized in that it comprisesthe implementation of at least one probe specific for at least onesequence of said human Liv21 complex according to claim
 2. 8. Method ofdetecting the human complex Liv21 according to claim 6, furthercomprising the implementation of at least one probe specific for atleast one of said complex sequence of these Liv21 human complex andassociated partners according to claim
 3. 9. Method of detecting thehuman complex Liv21 according to claim 6, further comprising theimplementation of at least one probe specific for at least one of saidcomplex sequence of these Liv21 human complex and associated partnersaccording to claim
 4. 10. Detection method according to claim 5,comprises at least the following steps: A step of extracting biologicalmaterial from a biological sample taken from a patient, A step ofcontacting said biological material with at least said specific probe ofany one of said sequences of said human complex Liv21 or human Liv21complex and its content partners, and at least one control and A step ofdetecting the expression products of gene expression said complex Liv21or said human Liv21 complex and its associated partners, said productsbeing comprised of expression of messenger RNA, or peptides, orproteins.
 11. Method detection according to claim 10, wherein screeninga candidate compound is capable of modulating the activity of said humancomplex Liv21, and in that it further comprises: a step of contactingsaid biological material with said candidate compound, and a step ofselecting said candidate compound.
 12. Method according to claim 11,wherein said biological sample is taken from a cancer patient and in ahealthy patient, and in that said material comprises nuclear cellextracts, cell extracts and cytoplasmic or cell extracts membrane, andin that it further comprises a step of determining the sub-expressionand overexpression of the gene products of said complex or said complexLiv21 or said Liv21 human complex and associated partners saidbiological extracts.
 13. Method according to claim 12, and furthercomprises a step of determining said radio sub-expression andoverexpression of said gene products: cell nuclear extracts andcytoplasmic cell extracts, cell extracts or membrane, and that itfurther comprises a step of analyzing combined radio said biologicalmaterial taken from a cancer patient and in a healthy patient
 14. Methodaccording to any one of claim 10, characterized in that it uses abiochip on which is deposited at least one sequence-specific probes fromsaid complex Liv21 human according to claim
 1. 15. Method according toclaim 10, characterized in that it applies to brain cancer,neuroblastoma, glioblastoma, without being limited there and breastcancer, bladder, skin and prostate and Alzheimer disease.
 16. Method oftreating cancers using an siRNA selected from siRNA SEQ ID NO: 91 to118.
 17. Method of treating cancers by injection of a peptide selectedfrom a vectorized peptide of SEQ ID No: 215 to 220.